Insulin-like growth factor binding protein -2 and -4 messenger ribonucleic acid expression in bovine ovarian follicles: effect of gonadotropins and developmental status

This work is concerned with the role of insulin-like growth factor binding protein (IGFBP)-2 and -4 in the regulation of IGF bioactivity in bovine follicles during the development of dominance. We measured the expression of IGFBP-2 and -4 messenger RNA (mRNA) in small (1-4 mm) gonadotropin-sensitive follicles and medium (4-8 mm) and large (>8 mm) gonadotropin-dependent follicles using in situ hybridization. In healthy nonatretic bovine follicles, IGFBP-2 and -4 mRNA expression was confined to granulosa and theca tissue, respectively. Moreover, during the development of follicular atresia, there were distinct changes in the temporal and spatial expression of these genes. IGFBP-2 immunoactivity was localized in granulosa tissue and the basement membrane of healthy preantral follicles, whereas IGFBP-4 immunoactivity was localized in both theca and granulosa tissue. Of particular interest was the lack of IGFBP-2 mRNA expression in large (>8 mm) gonadotropin-dependent follicles, an observation that was confirmed by the lack of immunoreactive IGFBP-2 in these follicles. The regulation of IGFBP-2 and -4 mRNA expression in granulosa and theca cells was analyzed using a serum-free cell culture system. FSH inhibited the expression of IGFBP-2 mRNA in granulosa cells, whereas LH stimulated IGFBP-4 mRNA expression in theca cells. Our results provide evidence for the existence of different roles for IGFBP-2 and -4 in the developing follicle.     

Cyclic changes of vasculature and vascular phenotypes in normal human ovaries

In order to study alterations of angiogenesis and blood vessel regression through ovarian cycle in human ovaries we quantitatively examined vascularity in various stages in 24 normal human ovaries. Vascular density (VD; vessel numbers/10(-7) m2) and endothelial area of each vessel (EA; 10(-12) m2/vessel) were evaluated using immunohistochemistry of CD34 and CAS 200 image analysis system. Small-sized vessels were sporadically observed in stroma adjacent to primordial or primary follicles (6.73 +/- 1.83 for VD and 113.58 +/- 21.80 for EA). Formation of capillary network was observed in the theca layer of preantral follicles (PA; 15.28 +/- 2.77 for VD and 113.58 +/- 21.80 for EA), and higher density of the capillary network was detected in non-dominant follicles in follicular phase (ND-F) and dominant follicles (DF; 29.33 +/- 3.84 for VD and 179.69 +/- 41.25 for EA). Dense capillary network was still present in non-dominant follicles in luteal phase (ND-L) and atretic follicles (AF; 26.88 +/- 3.36 for VD and 110.88 +/- 50.53 for EA). After ovulation, developing capillaries were also observed in the luteinized granulosa layers in early corpus luteum (21.95 +/- 2.06 for VD and 167.08 +/- 29.59 for EA). Vessel density markedly increased in mid corpus luteum, reached plateau in late corpus luteum (60.85 +/- 5.92 for VD and 70.99 +/- 15.57 for EA) and remained constant during degenerating corpora lutea. Vascular endothelial growth factor was immunohistochemically observed in the theca cells in PA, ND-F, DF and ND-L in follicular stages, and functioning corpora lutea. Immunoreactivity of intercellular adhesion molecule-1 was detected only in post-capillary venules in early degenerating corpora lutea. These findings suggest that ovarian angiogenesis is a requirement for the early stages of folliculogenesis and luteal growth, and also plays an important role in the process of follicular atresia and luteal regression.     

The mouse intraovarian insulin-like growth factor I system: departures from the rat paradigm

Although the rat intraovarian insulin-like growth factor I (IGF-I) system is well documented, the increasing availability of null mouse mutants for components of the IGF system necessitates characterization of the mouse model as well. Therefore, we undertook to define the components of the mouse intraovarian IGF-I system and to examine its operational characteristics. The cellular pattern of ovarian gene expression was comparable in the immature rat and mouse for IGF-I and the type I IGF receptor. In both species, IGF-I messenger RNA (mRNA) is selectively expressed by granulosa cells in growing, healthy appearing follicles. Type I IGF receptor mRNA was also concentrated in granulosa cells, but was uniformly expressed in all follicles large and small, healthy and atretic appearing alike. Cellular patterns of IGF-binding protein (IGFBP) gene expression were similar in mouse and rat, except in the case of IGFBP-2. IGFBP-2 mRNA was localized to the mouse granulosa cell, in contrast to its concentration in the rat thecal-interstitial compartment. This difference in IGFBP expression pattern was also noted in cultured mouse and rat granulosa cells. Although immunoreactive IGFBP-4 (24 and 28 kDa) and IGFBP-5 (29 kDa) were shared by both species, the cultured mouse granulosa cell also featured immunoreactive IGFBP-2 (30 kDa). The mouse paradigm further differed from its rat counterpart in that a maximal dose of FSH, previously shown to suppress the elaboration of rat granulosa cell-derived IGFBPs, was without effect. The addition of IGF-I proved stimulatory to the accumulation of the 28- to 29-kDa IGFBPs, as previously reported for the rat. However, IGF-I proved inhibitory to the accumulation of the 24-kDa IGFBP (presumptive nonglycosylated IGFBP-4); no consistent effect was reported for the rat model. Functional comparisons of mouse and rat ovarian cell cultures revealed qualitatively comparable FSH-stimulated steroidogenesis, disposition of radiolabeled pregnenolone, IGF-I-amplified FSH action, and IGFBP-mediated antigonadotropic activity. These findings indicate that the mouse intrafollicular IGF-I system differs from the rat paradigm in both the makeup and regulation of granulosa cell-derived IGFBPs as well as in the intensity and character of the steroidogenic process. Studies employing the mouse model must take into account these important distinctions relative to the more established rat paradigm.     

Immunocytochemical comparison of cultured normal epithelial prostatic cells with prostatic tissue sections

The cytologic localization and cellular levels of myc oncoprotein in the human ovary during follicular growth, regression and atresia were examined by the avidin/biotin immunoperoxidase method with a specific antibody to myc oncoprotein. In primordial follicles, only the oocyte showed intense immunostaining for myc protein, whereas the granulosa cells were negative for the staining. In preantral follicles, both the oocyte and granulosa cells were moderately immunostained for myc protein. In antral and preovulatory follicles, there was no appreciable staining for myc protein in the granulosa or theca cells, while myc protein staining in the oocyte persisted with less intensity. It is of interest that myc protein expression in granulosa cells was apparent only during the preantral follicle stage. Corpora lutea during the early and mid luteal phase were negative for myc protein staining, whereas in regressing corpora lutea during the late luteal phase, peripheral theca lutein cells adjacent to the central core of scar tissue were immunostained for myc protein. Corpora albicans showed no staining for myc protein. In atretic follicles, granulosa cells and theca interna cells demonstrated positive staining for myc protein. Ovarian stromal cells were negative for the immunostaining throughout the menstrual cycle. This demonstrates that myc protein is expressed in a stage-limited manner in the human ovary during follicular growth and regression. The abundant expression of myc protein in the oocyte at the primordial and preantral follicle stages and in the granulosa cells at the preantral follicle stage suggests a role for myc expression in the initial growth of the oocyte as well as in the autonomous growth of granulosa cells during the preantral stage seemingly independent of gonadotropic stimulation. Furthermore, notable expression of myc protein in the granulosa cells and theca interna cells of atretic follicles and in the peripheral theca lutein cells of regressing corpora lutea implies the possible participation of myc expression in remodelling the ovarian local tissue following atresia and luteolysis in the human ovary.     

In vivo hormonal regulation of insulin-like growth factor binding protein-5 mRNA in the immature rat ovary

OBJECTIVE: Despite the potential importance of insulin-like growth factor binding protein-5 (IGFBP-5) to follicular development, the hormonal regulation of this antigonadotropic IGFBP has not been investigated. Therefore, it was the objective of this study to eludicate the role of gonadotropins and estrogen in the in vivo regulation of IGFBP-5 mRNA expression. METHODS: Two models of follicular development in immature rats were used. Specifically, rats were hypophysectomized and treated with FSH and/or diethylstilbestrol (DES). Alternatively, terminal follicular development was induced in intact immature rats by pregnant mare serum gonadotropin (PMSG) and hCG. The IGFBP-5 mRNA in whole ovarian RNA was assayed by Northern blot hybridization. Localization of expression in PMSG and hCG-stimulated ovaries was further assessed by in situ hybridization. RESULTS: Expression of IGFBP-5 mRNA was increased in ovaries from hypophysectomized rats. Treatment with FSH and/or DES did not alter the abundance of this mRNA. Treatment with PMSG induced a transient increase in IGFBP-5 expression that was localized in a subset of alpha-inhibin-negative follicles. At later times after PMSG, IGFBP-5 expression persisted in the surface epithelium but was not detected in large preovulatory follicles. In vitro studies affirmed the antigonadotropic action of IGFBP-5. CONCLUSION: In vivo expression of IGFBP-5 in the rat ovary is moderated by hormonal treatment both in terms of total expression and follicular localization.     

Expression of steroidogenic enzyme and gonadotropin receptor genes in bovine follicles during ovarian follicular waves: a review

Ovarian follicular development in cattle is characterized by waves of growth during the prepubertal and postpartum periods and during estrous cycles. Each wave of follicular growth is characterized by recruitment of a cohort of follicles 4 to 5 mm in diameter. From the cohort, one follicle is selected for continued growth and becomes dominant. If luteolysis occurs during the growth phase of dominant follicles, final maturation and ovulation occurs. If luteolysis does not occur during the growing and maintenance phase of follicles, the fate is atresia. Changes in mRNA expression for the gonadotropin receptors (FSHr and LHr), key steroidogenic enzymes (cytochrome P450 side chain cleavage [P450scc], cytochrome P450 17alpha-hydroxylase-[P450c17], cytochrome P450 aromatase [P450arom], and 3beta-hydroxysteroid dehydrogenase [3beta-HSD]), and growth factors (IGF-I and -II) and their binding proteins (IGFBP) have been associated with different stages of follicular growth and atresia. In general, expression of mRNA for the gonadotropin receptors, steroidogenic enzymes, and steroidogenic acute regulatory protein (StAR) increase with progressive follicular development and is highest when dominant follicles approach maximum size. Expression of mRNA declines rapidly and becomes low or undetectable in atretic follicles. The IGF-I (granulosal cells) and IGF-II (thecal cells) are increased, whereas IGFBP-2 (granulosal cells) is reduced, in dominant follicles. Recruitment of a cohort of follicles is associated with initiation of expression of mRNA for P450scc and P450arom in granulosal cells. Selection of dominant follicles is associated with expression of mRNA for LHr and 3beta-HSD in granulosal cells. Thus, changes in gene expression likely are important to recruitment, selection, dominance, and atresia in ovarian follicles.     

Calcium concretions in the pineal gland of aged rats: an ultrastructural and microanalytical study of their biogenesis

OBJECTIVE: We summarized general principles related to apoptosis and reviewed the current evidence regarding apoptosis in the normal ovary during follicular atresia, ovulation, and corpus luteum formation and regression, and in the polycystic ovary. DATA SOURCES: Published articles and abstracts from national scientific meetings were reviewed. DATA SYNTHESIS: Apoptosis is a physiologic type of cell death that may regulate tissue remodeling. In the ovary, apoptosis occurs in atretic follicles, in the ovarian epithelium at the time of ovulation, and in the corpus luteum. Certain apoptosis-related genes are expressed during follicular atresia, which suggests a role for genes in the regulation of apoptosis in the ovary. In a rat model of polycystic ovaries, apoptosis occurs in both atretric follicles and cysts. CONCLUSIONS: Apoptosis is an important physiologic process in the ovary, and abnormalities in this process may lead to pathologic conditions. The study of ovarian apoptosis is still at an early stage, and further research will uncover the steps in this process.     

Expression of messenger ribonucleic acid (mRNA) encoding 3beta-hydroxysteroid dehydrogenase delta4,delta5 isomerase (3beta-HSD) during recruitment and selection of bovine ovarian follicles: identification of dominant follicles by expression of 3beta-HSD mRNA within the granulosa cell layer

The objective of the present study was to examine changes in expression of mRNA encoding 3beta-hydroxysteroid dehydrogenase delta4,delta5 isomerase (3beta-HSD) during recruitment and selection of bovine ovarian follicles. Dairy heifers (4-5/time period) were ovariectomized at 12, 24, 36, 48, 60, 72, 84, or 96 h after initiation of the first follicular wave (Time 0) following estrus. Expression of 3beta-HSD mRNA was localized by in situ hybridization and quantified by image analysis. Expression of 3beta-HSD mRNA was first detected in theca interna cells of preantral follicles with a well-developed theca layer and in granulosa cells of follicles > or = 8 mm in diameter. Regardless of stage of follicular wave, expression of 3beta-HSD mRNA in granulosa cells of follicles > or = 8 mm was correlated with follicular size (r = 0.665; p < 0.01). The 36-h time period appeared to be a transition period for selection since dominant follicles were detected by size and expression of 3beta-HSD mRNA in some cows but not in others. By 48 h after wave initiation, dominant follicles could be identified by both size and expression of 3beta-HSD mRNA. Expression of mRNA for 3beta-HSD in theca cells was higher (p < 0.05) at 24 h than at 12 h and remained elevated thereafter through 96 h. In contrast to theca cells, expression of mRNA for 3beta-HSD was undetectable within granulosa cells at 12 and 24 h. At 36 h, 3beta-HSD mRNA was expressed in granulosa cells of healthy follicles > or = 8 mm, and expression was higher (p < 0.05) at 48 h compared with 36 h. Expression of 3beta-HSD mRNA levels increased further in granulosa cells (p < 0.05) at 84 and 96 h compared to 48 h. Upon detection of mRNA for 3beta-HSD in granulosa cells, high levels of expression were always found in one (dominant) follicle/cow with the exception of two cows at 36 and 84 h that expressed 3beta-HSD mRNA in two large healthy follicles. Expression of 3beta-HSD mRNA was also detectable in granulosa cells of a few large atretic follicles in which remnant granulosa cells appeared to be luteinized. Healthy follicles expressed higher (p < 0.05) levels of 3beta-HSD mRNA in both theca and granulosa cells than did atretic follicles. Expression of 3beta-HSD mRNA in theca cells was higher (p < 0.01) in dominant follicles than in other subordinate healthy follicles. These results indicate that only selected dominant follicles express 3beta-HSD mRNA within granulosa cells, and expression increased in both thecal and granulosa cells during the follicular wave. Therefore, expression of 3beta-HSD mRNA within granulosa cells may be associated with the mechanism of selection of the dominant follicle during a follicular wave and may be required for maximum steroid production during follicular dominance.     

Circulating and ovarian IGF binding proteins: potential roles in normo-ovulatory cycles and in polycystic ovarian syndrome

IGFs function as co-gonadotropins in the ovary, facilitating steroidogenesis and follicle growth. IGFBP-1 to -5 are expressed in human ovary and mostly inhibit IGF action in in vitro ovarian cell culture systems. In the clinical disorder of polycystic ovarian syndrome (PCOS), which is characterized by hyperandrogenemia, polycystic ovaries and anovulation, follicles have a higher androgen: estradiol (A : E2) content and growth is arrested at the small antral stage. In the PCOS follicle, follicle stimulating hormone (FSH) and IGF levels are in the physiologic range, and even in the face of abundant androstenedione (AD) substrate, aromatase activity and E2 production are low. When PCOS granulosa are removed from their ovarian environment, they respond normally or hyperrespond to FSH. It has been postulated that an inhibitor of IGF's synergistic actions with FSH on aromatase activity may be one (or more) of the IGFBPs, which contributes to the arrested state of follicular development commonly observed in this disorder. High levels of IGFBP-2 and IGFBP-4 are present in follicular fluid (FF) from androgen-dominant follicles (FFa) from normally cycling women and in women with PCOS. This is in marked contrast to the near absence of these IGFBPs in estrogen-dominant FF (FFe), determined by Western ligand blotting. Regulation of granulosa-derived IGFBPs is effected by gonadotropins and insulin-like peptides. In addition, an IGFBP-4 metallo-serine protease is present in FFe, but not in FFa in ovaries from normally cycling women and those with PCOS, although the IGFBP-4 protease is present in PCOS follicles hyperstimulated for in vitro fertilization. Recent studies demonstrate that IGF-II in FFe is higher than in FFa' whereas IGF-I, IGFBP-3 and IGFBP-1 levels do not differ, underscoring the importance of local IGF-II production by the granulosa and the importance of IGFBP-4 and IGFBP-2 in regulation of IGF-II action within the follicle during its developmental pathway as an E2- or A-dominant follicle. In the androgen-treated female-to-male transsexual (TSX) model for PCOS, IGF-I, IGF-II, IGFBP-3 and IGFBP-1 levels do not differ.     

Characterization of the gene cassette required for biosynthesis of the (alpha1-->6)-linked N-acetyl-D-mannosamine-1-phosphate capsule of serogroup A Neisseria meningitidis

Apoptosis, the cellular mechanism of ovarian follicular atresia and luteal regression, is triggered by the activation of a proteolytic cascade of cysteine aspartate-specific proteases (caspases). The principle downstream effector of cell death is caspase-3, but little is known about the role or regulation of this enzyme in ovarian apoptosis. Two substrates of caspase-3, actin and poly(ADP-ribose) polymerase (PARP), are inhibitors of DNase I, which is the endonuclease responsible for ovarian apoptotic DNA degradation. We therefore investigated the proteolytic cleavage of actin and PARP as well as the localization of caspase-3 during follicular atresia (induced by gonadotropin withdrawal) and luteal regression (induced by prostaglandin F2alpha) in the rat ovary. Apoptotic DNA degradation was evident during both follicular atresia and luteal regression, but cleavage of PARP and actin was observed only during luteal regression. Caspase-3 was localized in luteal cells of healthy corpora lutea (CL) and in theca, but not in granulosa cells of healthy follicles. However, caspase-3 immunostaining was evident in granulosa cells of atretic follicles in a pattern similar to that of the localization of granulosa cell death. There was no difference between healthy and apoptotic CL in the distribution or intensity of caspase-3 staining. These results demonstrate that the cleavage of actin and PARP are not necessary for activation of apoptotic DNA degradation during ovarian apoptosis. In addition, the presence of caspase-3 in granulosa cells of atretic, but not healthy, follicles suggests that the expression of this enzyme is regulated by gonadotropin and may be up-regulated as part of the apoptotic process in granulosa cells.     

The ovarian renin-angiotensin system in reproductive physiology

The identification of the presence of prorenin, renin, angiotensinogen, angiotensin-converting enzyme, angiotensin II (Ang II), and Ang II receptors in the ovary suggests that there is a functional ovarian renin-angiotensin system (RAS). It could play a significant role in such areas of ovarian physiology as follicular development, steroidogenesis, oocyte maturation, ovulation, and follicle atresia. Expression of the ovarian RAS is regulated by gonadotropins. Ang II, a bioactive octapeptide of RAS, has important effects as a paracrine/autocrine regulator at different stages of the reproductive cycle. Ang II modulates ovarian steroidogenesis and formation of the corpus luteum and also stimulates oocyte maturation and ovulation via Ang II receptors on granulosa cells. In addition, increasing evidence demonstrates that Ang II is a major factor in regulating the function of atretic follicles. In any physiologic system, aberrations result in the development of pathologic states. Disturbances in the ovarian RAS can be the cause or the result of such reproductive disorders as polycystic ovary syndrome, ovarian hyperstimulation syndrome, ovarian tumors, and ectopic pregnancy. Data support the concept of an active and regulated RAS in ovarian follicles. Species differences observed in the expression of ovarian RAS suggest varying functional roles among species with respect to ovarian physiology.     

In situ hybridization of high density lipoprotein (scavenger, type 1) receptor messenger ribonucleic acid (mRNA) during folliculogenesis and luteinization: evidence for mRNA expression and induction by human chorionic gonadotropin specifically in cell types that use cholesterol for steroidogenesis

The present studies were undertaken to examine the expression of the high density lipoprotein (HDL) receptor, SR-B1 messenger RNA (mRNA) in ovarian cell types during folliculogenesis and luteinization using in situ hybridization histochemistry and to examine its hormonal regulation using Northern blots. For the in situ study for HDL receptor mRNA localization, 21-day-old rats were treated with 50 IU PMSG, and ovaries were collected 0, 24, and 56 h postinjection. At 56 h, animals were treated with a single dose of hCG, and ovaries were subsequently collected at 6-, 12-, 24-, and 72-h and 5-day intervals. In addition, on day 4 of pseudopregnancy, a second dose of 50 IU hCG or saline was administered, and ovaries were collected at 12, 24, and 48 h to determine the induction of the expression of HDL receptor mRNA. The results of in situ hybridization histochemistry showed that in the immature ovary, HDL receptor mRNA is associated with theca interna and interstitial cells (stroma). The mRNA expression in these cell types increased with PMSG treatment, but no signal was detected in the granulosa cells. Northern blot analysis also showed a marked increase in mRNA content in thecal and interstitial cells during follicular development. During luteinization, the intensity of the signal began to appear in the luteinized granulosa cells. With the completion of luteinization, the signal in the corpus luteum tissue became more intense. Further treatment with hCG increased the HDL receptor mRNA content compared with that in the saline-treated control. These results demonstrate that the cholesterol-using cell types of the ovary, namely the interstitial cells, thecal cells, and fully luteinized granulosa cells are endowed with the HDL receptor mRNA, which provides credence to the functional significance of the role of HDL receptor SR-B1 in cholesterol transport and ovarian steroidogenesis.     

How does daily treatment with human chorionic gonadotropin induce superovulation in the cyclic hamster?

Daily s.c. injection of 2.0 IU hCG per day, begun on Day 1 of the cycle (estrus), results in hamsters ovulating 20.7 +/- 0.7 eggs instead of the normal number of 13.3 +/- 0.5 (SEM). This is associated with a reduced rate of follicular atresia so that more of the 10 developing follicles per ovary (large preantral stages) normally recruited on Day 1 of the cycle mature and go on to ovulate. The hCG-treated follicles were larger than control follicles, but contained similar amounts of DNA/follicle; increased size of the antral cavity accounted for their greater size. Moreover, DNA synthesis was significantly reduced in the hCG follicles on Days 2 and 4. Thecal vascularity as judged by the number of red blood cells retained in the theca or microsphere uptake by follicles indicates that on Day 2, thecal blood flow was significantly lower in the hCG-treated animals than in controls. On the other hand, after hCG treatment begun on Day 1, serum levels and in vitro incubation of individual follicles revealed that on Day 2 and beyond, androstenedione (A) and estradiol (E2) levels were elevated. After hCG treatment, the elevated serum E2 correlated with reduced serum LH on Days 3 and 4 whereas FSH was unaffected. To study in vitro steroid accumulation, the 10 largest follicles (the developing follicles) were dissected from alternate left and right ovaries from control and hCG-treated animals and incubated individually, and their histology was then compared with the steroid profiles. Accumulation of A and E2 was significantly greater in the hCG-treated follicles than in controls in a 1-h basal incubation and after the addition of 50 ng LH. Progesterone accumulation usually did not differ between the control and hCG-treated follicles. Early stage 1 atretic follicles (judged by histology) were still capable of producing A and E2 in vitro, comparable to control follicles; but, as atresia progressed, the follicles synthesized only progesterone. This is consistent with the temporal pattern previously observed in a model of induced follicular atresia in the hamster [Greenwald, Biol Reprod 1989; 40:175-181]. It is concluded that superovulation resulting from hCG injections is due to thecal production of androgens from follicles normally destined for atresia. For the untreated cyclic hamster, the critical time for thecal androgen production is the first 2 days of the cycle. The aromatizable androgens are then converted into estrogens, which in turn may maintain the microenvironment of the antral cavity, which is essential for viability of the granulosa cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

The potential roles of intraovarian peptides in normal and abnormal mechanisms of reproductive physiology

Normal and abnormal follicular growth and steroidogenesis depend on gonadotropins as well as intraovarian peptide and polypeptide growth factors, which may mediate or potentiate gonadotropin action. Epidermal growth factor is mitogenic to ovarian granulosa and is a potent inhibitor of granulosa aromatase. It may be involved in the apparent arrest of follicular development commonly seen in women with polycystic ovarian syndrome as well as in the blunted response to gonadotropins seen in this syndrome. Insulin-like growth factors are also mitogenic to ovarian granulosa, but in contrast to epidermal growth factor, insulin-like growth factor-I, both alone and in synergy with gonadotropins, is a potent stimulus of aromatase and granulosa estradiol production. Insulin-like growth factor binding proteins-2 and -4, known inhibitors of insulin-like growth factor action, are higher in follicular fluid from atretic and polycystic ovarian syndrome follicles compared with estrogenic follicles and may be inhibitors of gonadotropin action in follicle selection and in polycystic ovarian syndrome. Cytokines including interleukins, tumor necrosis factor-alpha and interferon-gamma also appear to play a role in modulating ovarian steroidogenesis. Activins, inhibins, and follistatin (activin-binding protein) also affect follicular development and steroidogenesis and may play a role in dominant follicle selection and follicular atresia.     

Expression of androgen receptors and steroidogenic enzymes in relation to follicular growth and atresia following ovulation in pigs

Among the new antral follicles that develop after ovulation in pigs, the incidence of atresia, based on granulosa cell apoptosis, increases between Days 5 and 7 of the estrous cycle. The purpose of this study was to determine how follicular growth and atresia affected the expression of some key enzymes regulating follicular steroidogenesis and androgen receptor on Days 3, 5, and 7 after the onset of estrus. Ovaries were frozen in liquid propane for subsequent sectioning and immunohistochemical analysis. Ninety-six follicles were classified according to size as small (< 3 mm), medium (3-5 mm), or large (> 5 mm). Follicles in the active stages of the cell cycle were identified by the presence of the cell proliferation-associated nuclear antigen Ki-67 in granulosa cells. Follicles with apoptotic cells were identified by in situ 3'-end labeling of DNA. Staining intensity of antigens on sections was assigned a numeric value (0-3). Follicles assigned a value > 1 for 3'-end labeling in their granulosa cells were classified atretic. The percentage of atretic follicles increased (p < or = 0.05) from 5% on Days 3 and 5 to 41% on Day 7. Expression of Ki-67 in granulosa cells was more strongly (p < or = 0.05) associated with nonatretic follicles (98% expressing) than with atretic follicles (41% expressing). Aromatase cytochrome P450 (P450arom) was localized predominantly in granulosa cells of nonatretic follicles and was undetectable in atretic follicles. Androgen receptor in granulosa cells and expression of P450 17 alpha-hydroxylase/C17-20 lyase (P450c17) in theca interna were lower (p < or = 0.001) in atretic follicles than in nonatretic follicles. The expression of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) was localized to the theca interna and was unaffected by follicle atresia. In nonatretic small follicles, the expression of P450arom and P450c17 decreased (p < 0.01) between Days 3 and 7 while expression of Ki-67 was unchanged. In nonatretic follicles, increased follicle size was associated with a decrease (p < 0.01) in androgen receptor expression and increases (p < 0.01) in P450arom, P450c17, and 3 beta HSD expression. In conclusion, increased expression of steroidogenic enzymes was associated with follicular growth. Loss of P450arom expression in vivo is an early event in atresia and is followed by decreased cell proliferation, and decreased expression of androgen receptor and P450c17.     

Changes in extracellular matrix components and steroidogenic enzymes during growth and atresia of antral ovarian follicles in the sheep

To investigate the involvement of extracellular matrix (ECM) in folliculogenesis in the sheep, parallel changes in ECM components and key steroidogenic enzymes were studied by quantitative immunohistochemistry and immunoblotting during follicular growth and atresia. Growth of ovarian follicles from 1 to 5 mm in diameter was characterized by a progressive increase in P450 cholesterol sidechain cleavage levels in both thecal (p < 0.001) and granulosa cells (p < 0.001), an increase in P450 aromatase levels in granulosa cells of follicles larger than 3.5 mm (p < 0.001), and an increase in levels of P450 17 alpha-hydroxylase C17,20 lyase (P450(17 alpha)) in the theca interna. In addition, during follicular growth, a change in localization of cells expressing P450(17 alpha) within the theca interna was observed, positive cells being sparse within the theca interna of small follicles and specifically located close to the basal laminae in large follicles. In parallel, follicular growth was associated with an increase in levels of type I collagen in granulosa cell layers (p < 0.01) and an increase in levels of fibronectin (p < 0.05), particularly the specific ED-A alternatively spliced variant of fibronectin, in the theca externa. Follicular atresia was characterized by a loss of P450 aromatase in granulosa cells (p < 0.001) and a decrease in levels of P450(17 alpha) in the theca interna (p < 0.05). Simultaneously, levels of fibronectin (p < 0.05), particularly the ED-A variant of fibronectin, decreased in the theca externa of atretic follicles. Within the wall of granulosa cells, levels of fibronectin (p < 0.05), laminin, type IV collagen, and heparan sulfate proteoglycans strongly increased during follicular atresia. Overall, these results show that follicular growth and atresia were associated with distinct changes in levels of ECM components, suggesting that ECM components may play a role in the regulation of proliferation, differentiation, and apoptosis of follicular cells.     

Differential localization of laminin chains in bovine follicles

The composition of a basal lamina markedly affects its ability to filter material and affects the fate of adjacent epithelial cells. Therefore, basal laminae differ in composition with tissue development, and between different tissues in the body. Laminins are a component of basal laminae and consist of one alpha, one beta and one gamma chain, of which there are at least five, three and two isoforms, respectively. This is the first study to immunolocalize a range of these individual laminin chains (alpha 1, alpha 2, beta 1, beta 2, gamma 1) in ovarian follicles. Frozen sections of bovine ovaries (n = 6) were immunostained using specific antisera to laminin chains and factor VIII-related antigen (to identify endothelial cells). Secondary antisera were labelled with one of two different fluorochromes (DTAF and Cy3), and dual localization of laminin chains and factor VIII-related antigen was performed. The alpha 1, beta 2 and gamma 1 chains were consistently localized to the follicular basal lamina in all healthy follicles. Staining was less intense in the atretic antral follicles. Conversely, alpha 2 and beta 1 were rarely present in the follicular basal laminae of healthy antral follicles. Two of nine healthy antral follicles observed stained weakly for alpha 2 in their basal lamina, and beta 1 was present at low concentrations in growing preantral follicles. In atretic antral follicles, the follicular basal lamina stained positively for alpha 1, alpha 2, and beta 2 but no beta 1 was detected and the gamma 1 staining was less intense than in healthy follicles. Antisera to Englebreth Holm-Swarm tumour laminin stained basal laminae of all follicles. In the theca of antral follicles, beta 1 and beta 2 chains were both present in the vasculature. Staining for the gamma 1 chain was present in the thecal vasculature and generally throughout the theca of healthy and atretic antral follicles. Therefore, the composition of the follicular basal lamina alters during development and atresia, and potentially plays a role in the changing identity of the granulosa cells and the accumulation of antral follicular fluid.     

Mechanism, regulation, and manipulations of follicular atresia

In ovaries of mammals, an intense loss of germinal cells occurs by follicular atresia throughout the life. In atretic antral follicles, granulosa cells stop proliferating and become apoptotic. Main effectors of apoptosis are caspases which are activated by two ways in granulosa cells, the one involving Fas/TNF-alpha receptor, the other involving factors of the bel-2 family. Atresia is triggered when some essential factors supporting follicular development are lacking. Particularly, terminal follicular development is strictly dependent upon gonadotropin (FSH, then LH in the final preovulatory stage) supply, but factors acting in a paracrine way (growth factors, cytokines, steroids, constituents of extracellular matrix) play also important roles in amplifying gonadotropin action in follicular cells. Some pathological situations such as premature ovarian failure would result from accelerated follicular atresia, triggered by interactions between follicular cells and cells of the immune system. Current methods to control atresia consist in administrating exogenous gonadotropins, or indirectly increasing endogenous gonadotropins, or increasing follicular cell responsiveness to gonadotropins.     

The effect of LH, FSH and pregnant mares' serum gonadotrophin on ornithine decarboxylase activity in thecal and granulosa tissue during follicular growth and atresia in laying hens (Gallus domesticus)

The effect of ovine LH, porcine FSH and pregnant mares' serum gonadotrophin (PMSG) on the activity of ornithine decarboxylase activity in theca and granulosa tissue during folliculogenesis in laying hens is described. The changes in the activity of ornithine decarboxylase induced by hormonal challenge was used to measure the sensitivity of the tissue to the hormone. Thecal tissue from small (< 6 mm) follicles showed a large increase in the activity of ornithine decarboxylase 3 h after treatment with LH, FSH and PMSG, in vivo, whereas ornithine decarboxylase activity in thecal tissue from large (> 8 mm) preovulatory follicles and atretic follicles did not respond to any of the hormonal treatments. Ornithine decarboxylase activity in granulosa tissue from the largest preovulatory follicle increased significantly 3 h after treatment with LH and PMSG in vivo; no effect was observed with FSH. Granulosa tissue from the third largest and fifth largest preovulatory follicles were refractory to the hormonal treatments. Basal activity of ornithine decarboxylase in granulosa tissue from preovulatory follicles increased as the follicles approached ovulation, whereas the activity in thecal tissue from the same follicles decreased. The difference in sensitivity of thecal tissue from small and large preovulatory follicles towards gonadotrophin treatment in vivo is correlated with the difference in the observed rate of atresia occurring within the two groups of follicles. Atresia is the common fate for small follicles, whereas it is a rare event for large preovulatory follicles under normal physiological conditions.(ABSTRACT TRUNCATED AT 250 WORDS)