Early Stages Of Atresia Research Articles

Ultrastructural features of atretic ovarian follicles in buffalo (Bubalus bubalis)

The process of follicular development and features of morphological changes are essential to understand the factors involved during folliculogenesis and to improve fertility in buffalo. Hence the objective of the present study was to report ultrastructural features of atretic ovarian follicles in buffalo. In the early stage of atresia, the involvement of mitochondria was observed and whereas in the later stages of atresia, involvement of macrophages were observed. The granulosa cells of buffalo atretic tertiary follicles were observed to contain undulation of the nuclear membrane, crescent formation of the pyknotic nuclei and loopiness and undulations of the basal lamina. These features are similar to the reports in other species. The involvement of mitochondria in the early stage of atresia suggested the probable role of a reactive oxygen free radical induced damage.

Ultrastructural Features of Atretic Follicles in the Sexually Immature Ostrich (Struthio Camelus)

The aim of this study was to describe the ultrastructural features of atresia in follicles of the immature ostrich (12-14 months old); a ratite that displays seasonal, precocious ovarian activity. The early stage of atresia in primordial, pre-vitellogenic and vitellogenic follicles was characterized by the accumulation of lipid droplets in the granulosa cells. Granulosa cells with condensed cytoplasm and nuclei were a prominent feature during the intermediate phase of atresia. The degenerating follicles were then infiltrated by stroma during the terminal stages of atresia. The results of this study provide further information on the morphology of atretic ovarian follicles in the immature ostrich.

Gene expression for LH receptor, 17α-hydroxylase and StAR in the theca interna of preantral and early antral follicles in the bovine ovary

The onset of gene expression for three proteins that play pivotal roles in theca interna function, namely the LH receptor (LH-R), cytochrome P450 17 alpha-hydroxylase (17 alpha OH) and the steroidogenic acute regulatory protein (StAR), was determined. Ovaries were obtained on day 9 of the oestrus cycle from mature synchronized dairy cows (n = 5) and gene expression in preantral and antral follicles up to 4 mm in diameter was evaluated by in situ hybridization. LH-R and 17 alpha OH mRNAs were observed first, in the theca interna of large preantral follicles (type 4), concurrent with its morphological differentiation. StAR mRNA appeared later during follicular growth, in follicles >1 mm in diameter (type 6). LH-R and 17 alpha OH mRNAs were found exclusively in the thecal cells, whereas StAR mRNA appeared in thecal cells, granulosa cells of late atretic follicles and oocytes. In early atresia, thecal cells expressed all three mRNAs, and their expression decreased gradually as atresia progressed. Atresia in granulosa cells was characterized by massive apoptosis of periantral, but not peribasal cells, that differentiated into luteal-like cells expressing StAR. In summary, our study suggests that in spite of the presence of 17 alpha OH, a key enzyme in steroidogenesis, the ability to produce steroids by bovine follicles smaller than 1 mm in diameter must be very limited due to the absence of StAR protein. During the early stages of atresia, thecal cells remain morphologically and functionally healthy, and continue to express all three studied mRNAs.

222THE ROLE OF TUMOR NECROSIS FACTOR-RELATED APOPTOSIS INDUCING LIGAND DURING FOLLICULAR ATRESIA IN PIG OVARIES

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce cell death by binding to its receptors (DR4 and DR5). However, binding to DcR1 or DcR2 cannot induce apoptosis. DcRs compete with DRs. TRAIL has been reported to induce apoptosis in various tumor cells but not in normal cells. However, a recent study revealed that TRAIL induces apoptosis in normal hepatocytes of human but not in those of rat, mouse, or rhesus monkey, indicating that there are species-specific differences in TRAIL and receptor systems. In the present study, we demonstrated Immunohistochemical, Western immunoblotting, and reverse transcription-polymerase chain reaction analyses (RT-PCR) of TRAIL and DR4 in granulosa cells during follicular atresia in pig ovaries. For immunohistochemistry, pig ovaries obtained at a local slaughterhouse were fixed with 20% buffered formalin. For Western blotting and RT-PCR analysis, individual preovulatory antral follicles were dissected from the ovaries. Based on morphological and endocrinological criteria, the antral follicles were divided into three categories as follows: healthy, early stage of atresia, progressed stage of atresia. Significant increases were demonstrated in TRAIL protein and mRNA levels during atresia, but not in DR4 protein. Moreover in an in vitro apoptosis-inducing assay using cultured granulosa cells prepared from healthy follicles, we showed that more than 200ng/mL TRAIL could activate caspase-3 and induce apoptotic cell death in a dose-and time-dependent manner, but less than 100ng/mL of TRAIL could not induce apoptosis. When DcR1 was removed from the cell membrane of granulosa cells, a lower dose of TRAIL could induce apoptosis. The present findings suggested that the TRAIL can induce granulosa cell apoptosis, and that DcR1 blocks TRAIL-induced apoptosis in granulosa cells of healthy follicles in porcine ovaries.

Measurement of inhibin A and follicular status predict the response of ewes to superovulatory FSH treatments

Variability in superovulatory response to FSH stimulation is common to most mammals and imposes practical problems for assisted reproduction. In sheep, we have studied if this response is related to the ovarian follicular population and activity before the stimulation. During the breeding season, 30 ewes were treated with 40 mg FGA sponges for 14 days and 125 μg cloprostenol injection on Day 12, considering Day 0 as the day of progestagen insertion. Superovulatory response was induced with two different FSH regimes using the same total dose (8.8 mg), administered twice daily from 60 h before to 24 h after progestagen withdrawal. At the first FSH injection, all follicles ≥2 mm were observed by transrectal ultrasonography and plasma FSH and inhibin A levels were determined. The number of corpora lutea and the number of and viability of recovered embryos in response to the treatment were determined on Day 7 after sponge withdrawal. No significant differences were found between treatments. The total mean number of corpora lutea (11.5±1.2) and recovered embryos (7.9±1.1) were positively correlated ( P<0.05 and <0.01, respectively) with the number of small antral follicles (2–3 mm; 9.2±0.7) and inhibin A concentration (240±18 pg/ml; P<0.05 for corpora lutea and P<0.005 for recovered embryos) observed at the onset of the superovulatory treatment, which was also positively correlated with the number of viable embryos (5.8±0.9, P<0.005). In 18 ewes with follicles ≥6 mm prior to FSH treatment, the ovulation rate was unaffected but the number of embryos (6.1±0.9 versus 11.6±2; P<0.05) and their viability (4.5±0.8 versus 8.5±2; P<0.05) was reduced. The lower number of embryos produced when a large follicle is present suggest that a proportion of the smaller follicles are in early stages of atresia and the developmental competence of their oocyte is compromised.

Oocyte maturation.

The oocyte is dependent on granulosa cells to provide nutrients and regulatory signals. Granulosa cells must be at the appropriate stage of differentiation to initiate these signals and transmit them to the oocyte. Studies have shown that in vitro-matured oocytes from follicles in early stages of atresia are more competent to support embryonic development than those from actively growing follicles. The acquisition of developmental competence appears to occur prior to in vitro maturation and can be induced by gonadotropin-free coasting in vivo or postmortem ovary incubation in vitro. The acquisition of developmental competence is probably a common signaling or differentiation pathway that occurs in the oocyte and/or associated granulosa regardless of whether the oocyte is destined to ovulate or degenerate. Early follicle atresia is the visually discernible characteristic in vitro that is associated with increased developmental potential.

Characterization of calf follicular fluid and its ability to support cytoplasmic maturation of cow and calf oocytes.

The aims of the present study were to characterize the follicular fluid from prepubertal calf follicles of known size and quality and to study the ability of follicular fluid to support cytoplasmic maturation of calf and cow oocytes. Follicular fluid was obtained from 67 calf follicles classified according to size (S: small < 6 mm, M: medium 6-8 mm and L: large > 8 mm in diameter) and quality (HY: healthy, EA: early atretic and A: atretic). Quality was first determined by mitosis:pycnosis ratios in granulosa cell smears and confirmed by insulin-like growth factor binding protein (IGFBP) patterns. There was approximately 90% agreement between the two methods of follicle classification and on this basis the calf follicular fluid was pooled into nine groups. The accuracy of this pooling was confirmed by evaluation of oestradiol concentrations in the nine pools of follicular fluid using radio-immunoassay. Increases in follicle size were characterized by a decreased intensity of bands for IGFBP-2, IGFBP-5 and IGFBP-4, an increase in the proportion of healthy follicles and a decrease in the proportion of follicles in the early stages of atresia. This finding is in agreement with previously published results in cows. All classes of calf follicular fluid contained lower concentrations of oestradiol than previously reported for corresponding classes of cow follicular fluid. Cow oocytes were matured in M199 alone, or supplemented with 10% fetal calf serum (FCS), or 10% calf follicular fluid from one of three pools (LHY, LEA, LA), fertilized, and cultured for 8 days in synthetic oviduct fluid. Addition of FCS or calf follicular fluid to cow oocytes during in vitro maturation increased the yield of blastocysts on day 8 over the control (23%, 21/91), FCS (39%, 37/96, P < 0.05), LA (41% 21/52, P < 0.05), LEA (32%, 28/88), LHY (36%, 32/88), although not significantly in all cases. The rate of hatching of blastocysts was also improved: control (38%, 8/21), FCS (54%, 20/37), LA (62%, 13/21), LEA (75%, 21/28, P < 0.02), LHY (59% 19/32). In contrast, the addition of either FCS, calf follicular fluid or cow follicular fluid did not improve development of calf oocytes compared with the unsupplemented control. In conclusion, it is probable that serum and follicular fluid contain factors that stimulate the acquisition by oocytes, during maturation, of developmental competence and to which prepubertal oocytes are unable to respond. Specific receptors for these factors may develop only around puberty.

Relationship between expression of integrins and granulosa cell apoptosis in ovarian follicles of the marmoset ( Callithrix jacchus)

DNA fragmentation (apoptosis) was studied during the follicular, periovulatory and luteal phase in the marmoset monkey ovary by means of terminal transferase mediated in situ nick end labeling, and correlated with immunohistochemical localization of integrins (β1, α2 and α6 subunits). For this purpose a double-labeling technique was developed. During all phases, apoptosis of granulosa cells was exclusively restricted to tertiary follicles displaying advanced stages of atresia (as morphologically determined). During early stages of atresia in tertiary follicles, indicated by widened intercellular spaces, no apoptosis was seen. Staining intensities for integrins β1 and α6 were strong in intact primordial/primary, secondary and tertiary follicles. Integrin expression of granulosa cells was weak in atretic tertiary follicles but not in atretic primary or secondary follicles. Double labeling revealed that DNA fragmentation was solely found in granulosa cells of tertiary follicles displaying faint or absent staining for both integrin subunits. During the periovulatory and the luteal phase, granulosa cells of atretic tertiary follicles bordering on the basal membrane, which were referred to as luteinizing cells, expressed the β1 subunit as well as the α2-integrin subunit whereas granulosa cells neighboring to the antrum were apoptotic and negative for integrin immunoreactivity. In summary, early atresia of tertiary follicles is first characterized by morphological alterations as wide intercellular gaps, without any signs of granulosa cell apoptosis. Advanced stages of atresia, in tertiary follicles however, are accompanied by apoptosis of granulosa cells and a faint or absent staining for integrin subunits β1 and α6. According to recent in vitro findings, our results point to a possible relation between granulosa cell apoptosis of tertiary follicles and integrin expression in ovarian marmoset follicles.

Apoptosis Occurs in Granulosa Cells but not Cumulus Cells in the Atretic Graafian Follicles in Multiparous Pig Ovaries.

The process of apoptosis in granulosa and cumulus cells of ovarian follicles during follicular atresia was investigated in multiparous pigs. In healthy follicles, no apoptotic cells were observed histochemically, and no DNA ladder formation or Ca2+/Mg2+-dependent endonuclease activity were detected biochemically in granulosa or cumulus cells. In the follicles in the early stage of atresia, apoptosis occurred in granulosa cells located on the inner surface of the follicular wall but not cumulus cells. NA ladder formation was observed in granulosa but not cumulus cells, and high levels of endonuclease activity were demonstrated only in the granulosa cells. No endonuclease activity was detected in cumulus cells obtained from the same atretic follicles. These findings suggest a lack of apoptotic cell death in cumulus cells in atretic follicles in multiparous pig ovaries.

Immunological evidence for the expression of the Fas antigen in the infant and adult human ovary during follicular regression and atresia.

Immunohistochemical localization of the Fas antigen in the infant and adult human ovary during follicular growth, regression, and atresia was examined by the avidin/biotin immunoperoxidase method with a monoclonal antibody to the Fas antigen. Western blotting was used to confirm the presence of the Fas antigen protein. In primordial and primary follicles within the normal adult ovary, only the oocyte showed moderate immunostaining for the Fas antigen. In secondary and antral follicles, only the oocyte showed weak staining for the Fas antigen, while in preovulatory follicles, neither the oocyte nor the granulosa and theca cells were immunostained for the Fas antigen. In corpora lutea, the Fas antigen staining became apparent in the granulosa lutein cells during the early luteal phase and intensified during the mid luteal phase, while the theca lutein cells became positive for the Fas antigen staining during the mid luteal phase. During the late luteal phase, the staining intensity of the Fas antigen in the regressing corpora lutea further increased. As the regressing corpora lutea were converted into corpora albicans, the staining intensity decreased, and the corpora albicans and stromal cells were negative for the Fas antigen. In atretic primordial and primary follicles, only the degenerating oocyte showed the Fas antigen staining. By contrast, in atretic antral follicles, the Fas antigen staining was profound in the degenerating granulosa cells at the early stage of atresia, and at the mid stage of atresia it was intensified in the cell surface of the scattered granulosa cells and became apparent in the theca cells. At the late stage of atresia the Fas antigen remained only in the hypertrophied theca cells. In the infant ovary, only the oocyte in primordial and primary follicles exhibited intense staining for the Fas antigen. In the postmenopausal ovary, the Fas antigen staining was entirely negative. Western blot analysis revealed the presence of the Fas antigen protein with a molecular mass of 45 kDa in luteal tissues. On the basis of the recent evidence, that the Fas antigen mediates an apoptotic signal in a variety of cells, the abundant expression of the Fas antigen in the regressing corpora lutea and atretic follicles suggests that the Fas antigen participates in luteal regression and follicular atresia through the apoptotic process. Furthermore, notable expression of the Fas antigen in the oocyte of primordial and primary follicles within the infant and adult human ovary followed by the decrease in the Fas antigen expression in the oocyte with the advance of follicular maturation suggests that the Fas antigen expression in the oocyte may play a role in follicular selection.

Fas/APO-1/CD95 system as a mediator of granulosa cell apoptosis in ovarian follicle atresia.

Current studies have shown that atresia of ovarian follicles is induced through apoptosis in granulosa cells. Several articles have been devoted to study of the molecular mechanisms responsible for APO-1/CD95 (Fas) is a cell surface protein that can mediate apoptosis in lymphoid cells, and Fas ligand was recently identified in a cytotoxic T cell line. To clarify the involvement of the Fas-Fas ligand system in granulosa cell apoptosis, we investigated the expression of Fas and Fas ligand at an individual cell level. For this purpose, we raised specific polyclonal antibodies against Fas and Fas ligand. Western blotting confirmed that our anti-Fas antibodies (anti-P2 and anti-P4) detect a specific band with a mol wt of 45 kDa in the lysate of ovaries from immature PMSG-treated rats or adult cyclic rats. In immature PMSG-treated rats, immunohistochemical analysis with these antibodies revealed specific staining of granulosa cells in secondary and tertiary follicles at an early stage of atresia, but not in healthy follicles. Fas messenger RNA was also found in granulosa cells of early atretic follicles using in situ hybridization. On the other hand, the anti-Fas ligand antibody (anti-P5) detected a specific 31-kDa band on a Western blot of the oocytes lysate, and the staining with the serum was localized to oocytes in most of developing follicles. Colocalization of Fas and Fas ligand in certain follicles intimately correlated with granulosa cell apoptosis, which was revealed by terminal deoxynucleotidyl transferase-mediated deoxy-UTP-biotin nick end labeling staining of DNA strand breaks. Finally, we found that interferon-gamma increased Fas expression on granulosa cells in vitro. Coculturing interferon-gamma-pretreated granulosa cells with zona-free oocytes induced granulosa cell apoptosis, which was confirmed by Hoechst 33342 dye staining and terminal deoxynucleotidyl transferase-mediated deoxy-UTP-biotin nick end labeling, and the killing effect of oocytes was abolished by the addition of anti-P2, which was expected to interrupt the interaction between Fas and Fas ligand. These results demonstrate that activation between Fas and Fas ligand. These results demonstrate that activation of the Fas-Fas ligand system is capable of initiating apoptosis in the ovary, as are a number of other stimuli, outside the immune system.

Proteolytic and antiproteolytic activities in goat antral follicles

Goat antral follicles were dissected and classified initially by size into three groups: <3 mm, between 3 and 6 mm, and >6 mm. Follicles less than 3 mm diameter were studied without any further classification. All others were classified according to their gross stereoscopic microscope appearance into four sub-groups. Histological observation showed that sub-group 1 consisted mainly of non-atretic follicles, sub-group 2 of follicles in the early stages of atresia, while sub-groups 3a and 3b were formed by secondary and tertiary atretic follicles. Tryptic and anti-trypic activites were measured in follicular fluid, homogenized follicular cells and empty follicular sacs, using a fluorometric procedure. Antitryptic activity in follicular fluid was found to be directly correlated with progress of atresia and inversely with follicular growth, being smallest in big (>6 mm), non-atretic follicles and consistently higher in small (<3 mm) and in atretic follicles. No differences were detected between sub-group 3 follicles of different sizes. After elimination of anti-trypic activity by gel filtration, trypsin-like activity in cell-free follicular fluid was found only on >6 mm non-atretic follicles. High trypsin-like activity was found in the cell walls of non-atretic or early atretic follicles. This activity was significantly lower in sub-group 3a follicles and was almost absent in tertiary atretic follicles (sub-group 3b). In contrast, trypsin-like activity in granulosa cells recovered by centrifugation of follicular fluid was twice as high in sub-group 3a as in sub-groups 1 and 2 and almost five times higher than in sub-group 3b.

The three-dimensional structure of the zona pellucida in growing and atretic ovarian follicles of the mouse

The present study provides further details on the fine-structural three-dimensional architecture of the zona pellucida (ZP) in growing and atretic follicles of mice by use of ruthenium red in combination with the detergents Triton X100 and saponin. These detergents were used for extraction of the "soluble" fraction of the zonal proteins in an attempt to expose the "structural" zonal glycoproteins, which in turn can be viewed as minute three-dimensional networks upon transmission- and scanning electron-microscopic examination. By use of these methods, the ZP of growing follicles appeared to be formed by interconnected filaments which also bind to globular structures building up a three-dimensional lattice. In contrast, the ZP of stage I as well as other (II and III) stages of atretic follicles showed a structure characterized by the presence of closely packed granules connected with short filaments to form a close-mesh reticulum. This structural change of the ZP, which in the present study is also associated with the disappearance of "gap junctions" within the granulosa and cumulus cell population, might represent one of the early events involved in the onset of atresia. These changes, most probably depending on an altered secretory activity of both oocytes and follicle cells, might lead to a degradation of the ZP network structure and to its subsequent increased density (condensation). All these morphodynamic events eventually contribute to a sequestration of the oocyte in the early stage of atresia.

Enzyme histochemical observations on the ovarian small follicles in relation to the mechanism of atresia in laying hens (Gallus domesticus).

In order to examine the mechanism of atresia in the small follicles of laying hens, changes of three enzyme activities, acid phosphatase, 3β-hydroxysteroid dehydrogenase (3β-HSD) and glucose-6-phosphate dehydrogenase (G-6-PD), during atresia were analyzed histochemically. Acid phosphatase activity in the follicular wall increased along with the tissue degradation in the process of atresia. High activities of 3β-HSD and G-6-PD which were observed only in the thecal interstitial cells of small follicles did not decrease at the early stage of atresia, but decreased as atresia proceeded. It is suggested that lysosomal enzymes may be involved in the tissue degradation during atresia. It is perhaps premature to conclude that the decrease of the production activities of steroid hormones, whose synthesis depends on 3β-HSD and G-6-PD, in individual follicles is involved in the induction of atresia.

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.

Inhibitory effect of progesterone on follicular growth and induced superovulation in the rat.

We investigated the effect of exogenous progesterone on follicular growth and pregnant mares' serum gonadotropin and human chorionic gonadotropin (PMS-HCG) induced superovulation in intact and hypophysectomized immature rats. Although the administration of progesterone during PMS-HCG injections had no effect on superovulation, the administration of progesterone beginning 3 days before PMS injection inhibited superovulation in both intact and hypophysectomized rats. In order to study the mechanism of inhibition of superovulation by pretreatment with progesterone, an autoradiographic study using tritiated thymidine was done in hypophysectomized rats just before PMS injection. The labelling indices of granulosa cells were significantly decreased by the administration of progesterone (p < 0.001). Treatment with progesterone resulted in an increase of the follicles in early stages of atresia with pycnotic nuclei in granulosa cells surrounding apparently normal oocytes. Ovarian histology and serum and ovarian tissue concentrations of estradiol were then examined in hypophysectomized rats 20 h after HCG injection. Large Graafian follicles and corpora lutea were rarely seen in rats previously given progesterone, whereas many large Graafian follicles and corpora lutea were observed in the control rats. The concentrations of estradiol in the progesterone-pretreated rats were significantly reduced in serum (p < 0.02) and slightly reduced in ovarian tissues when compared with controls.

Studies on guinea pig oocytes. II. Histochemical observations on some phosphatases and lipid in developing and in atretic oocytes and follicles.

AbstractNormal guinea pig oocytes develop in an environment of AMPase within the granulosa cells of the wall, the cumulus and the latter's projections that traverse the zona pellucida to terminate at the surface of the oocyte. Oocytes in developing non‐vesicular and small vesicular follicles have an ATPase activity at their surface, which appears to indicate specialized absorptive mechanisms confined to these early stages of development. Acid phosphatase, present in granules scattered throughout the cytoplasm of all developing oocytes, may be evidence of a mechanism in this species, whereby the oocyte hydrolyzes unidentified nutritional reserves.Histologically normal appearing oocytes in atretic vesicular follicles may show deviations from the normal histochemical pattern, i.e., clumping and subsequent loss of acid phosphatase activity or a band of AMPase activity at the periphery of the oocyte representing apparent enlargement of the terminals of projections of the cumulus. These deviations may reflect disturbances of granulosa cell‐oocyte transfer as a consequence of beginning atetric processes in the follicle wall. By the eighth day of the estrous cycle the granulosa layer of many medium sized follicles has been invaded by a few long strands of nucleoside polyphosphatase positive thecal connective tissue often carrying a small blood vessel. This early stage of atresia is apparently followed by shedding of the granulosa layer and hypertrophy of the thecal layer which begins the transformation of atretic follicles into interstitial masses.