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.     

Prolactin concentrations in follicular fluid following ovarian hyperstimulation for in vitro fertilization

Prolactin (PRL) and sex steroid concentrations were measured in follicular fluids of women treated either with (1) clomiphene/hCG or with (2) clomiphene + hMG/hCG. Method 1 of ovarian stimulation resulted in lower follicular PRL and higher oestradiol-17 beta (E2) and progesterone (P) concentrations than method 2. There was no difference in the PRL and sex steroid concentrations of follicles with fertilized and of those yielding unfertilized ova, but in both stimulation types, follicles from which no oocytes were obtained had high PRL and low E2 and P levels. Significant positive correlations were evident for PRL and T and E2 and P, respectively, while PRL and P were negatively correlated.     

Influence of follicular maturation on luteinizing hormone and guanosine 5'-O-thiotriphosphate-promoted breakdown of phosphoinositides and calcium mobilization in chicken granulosa cells

Previous studies in this laboratory have shown a remarkable increase in the capacity of avian granulosa cells to produce progesterone during the last 2-3 days of folliculogenesis, with concomitant increases in the activities of key steroidogenic enzymes. In view of the proposed involvement of inositol phospholipids and their hydrolytic products in signal transduction in steroidogenic cells, we investigated in this study the influence of follicular maturation on phosphoinositide-specific phospholipase C (PLC) and intracellular Ca2+ release in chicken granulosa cells. Resting concentrations of intracellular calcium ([Ca2+]i) as measured in Fura 2/AM-loaded cells increased significantly during the last 48 h of follicular maturation, from 185 +/- 9 nM in the third largest follicle (F3) to 355 +/- 26 in the cells of the largest (F1) follicle. Luteinizing hormone (LH) caused a dose-related rise in [Ca2+]i, but the dose response was left-shifted by more than one order of magnitude in F1 cells compared to F2 cells. In granulosa cells of less developed follicles, LH failed to raise [Ca2+]i. To assess PLC activity, granulosa cells from F1, F2, and F3 follicles were labeled with [3H]inositol for 2 h and then stimulated with LH (0.1 microgram/ml). Time course studies showed that within 30 s, phosphatidylinositol-4,5 bisphosphate (PIP2) decreased by 33%, 13%, and 11% in F1, F2, and F3 cells, respectively. Similar responses were obtained when permeabilized cells were exposed to guanosine 5'-O-thiotriphosphate, which also caused a corresponding increase in 45Ca efflux from F1 and F2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of the environmental antiandrogen vinclozolin on the induction of granulosa cell apoptosis during follicular atresia in pigs

The aim of this study was to investigate whether the androgens testosterone and dihydrotestosterone (DHT) and the antiandrogenic fungicide vinclozolin (Vnz) exert proapoptotic effects on porcine granulosa cells (GCs), and to examine the roles of these compounds in follicular atresia. Granulosa cells isolated from pig follicles were cultured for 24 hours, and then exposed to 0.1 μM testosterone, 0.1 μM DHT, 14 μM Vnz, or the equivalent concentrations of testosterone and Vnz or DHT and Vnz for a further 24 hours. Apoptosis and necrosis of the GCs were determined via Hoechst staining and flow cytometry analyses of annexin V-stained cells. Whole porcine follicles were also exposed to the same compounds and combinations of compounds for 24 hours. The sections were stained with hematoxylin and eosin for morphologic assessments, and a Terminal deoxynucleotidyl Transferase Biotyn-dUTP Nick-End Labeling (TUNEL) assay was performed to determine the number of apoptotic cells. The progesterone and estradiol concentrations secreted into the culture media by isolated GCs and follicles were also measured. Exposure to the androgens resulted in an increased number of apoptotic GCs both in vitro and in the organotypic model. Vinclozolin exposure increased and decreased the number of necrotic and apoptotic GCs, respectively. Furthermore, compared with control follicles, those exposed to testosterone, DHT, or Vnz displayed enhanced atresia, and coadministration of Vnz attenuated the promotive effect of these androgens on atresia. Estradiol secretion was stimulated by the combination of testosterone and Vnz, whereas exposure to Vnz alone reduced it. Progesterone production declined after the combined addition of androgens and the antiandrogen. In summary, Vnz caused massive necrosis of GCs in vitro and induced apoptosis of GCs in whole follicles. The androgens testosterone and DHT enhanced these effects. The results presented here suggest that selective destruction of porcine follicles is a serious consequence of exposure to Vnz, and may lead to premature ovarian failure in affected animals.     

The physiological role of beta-endorphin in porcine ovarian follicles

Beta-endorphin-like immunoreactivity (beta-END-LI) was measured by radioimmunoassay in porcine ovarian follicular fluid (FF) from small, medium and large follicles throughout the oestrous cycle. The concentration of beta-END-LI in FF from small follicles collected on days 1-5 of the cycle was at least tenfold higher than in the fluid from any other follicles independently from their size and the period of the cycle. The level of beta-END-LI in small follicles on days 6-10 was drastically decreased. Subsequently, on days 11-16 its concentration was enhanced and reduced again in pre-ovulatory period of the cycle. Concentrations of beta-END-LI in FF from medium follicles were relatively equal throughout the cycle (days 6-21). No significant differences in beta-END-LI levels were found between small, medium and large follicles from days 17-21. However, beta-END-LI concentrations in medium follicles on days 11-13 and 14-16 were statistically lower than those in small follicles. Moreover, the effects of FSH, prolactin (PRL), progesterone (P4), testosterone (T) and 17 beta-oestradiol (E2) on beta-END-LI release by granulosa cells (GCs) from large follicles and, on the other hand, the effects of the opioid agonist FK 33-824 alone or in combination with FSH, PRL or naloxone (NAL) on follicular steroidogenesis were studied. FSH drastically increased beta-END-LI output in a dose-dependent fashion. This stimulatory effect of the gonadotrophin was inhibited by the highest dose of P4 (10(-5) M). The effect of PRL and the steroids added to the cultures on beta-END-LI release was negligible. FSH- or PRL-induced P4 secretion by GCs was essentially abolished by both FK 33-824 and NAL. However, androstenedione (A4) and testosterone output by the cells was greatly potentiated by FK 33-824. In the presence of NAL, FSH or PRL, A4 release stimulated by FK 33-824 was suppressed to the basal level. Secretion of E2 was completely free from the influence of FK 33-824 or NAL; only oestrone (E1) output was modulated by them in cultures where FSH or PRL was present. In conclusion, FSH appears to be the key regulator of beta-END-LI secretion by porcine granulosa cells. Moreover, steroidogenesis in pig granulosa cells is modulated by opioid peptides acting both alone and by way of interaction with FSH or PRL.     

Prolactin secretion in permeable GH3 pituitary cells is stimulated by Ca2+ and protein kinase C activators

We have used GH3 cells permeabilized by electric field discharge to examine the effects of Ca2+ and protein kinase C activators (phorbol ester and diacylglycerol) on prolactin (PRL) release. Ca2+ was found to stimulate PRL release approximately 4 fold at 3 microM Ca2+ with a half-maximal response at approximately .5 microM estimated free Ca2+. 12-O-tetradecanoyl phorbol-13-acetate and 1-oleoyl-2-acetyl-sn-glycerol stimulated PRL release throughout a range of Ca2+ concentrations (1 nM -3 microM), but stimulation was greater at higher Ca2+ concentrations (.1 microM to 1 microM). Both agents decreased by 1.8 fold the apparent [Ca2+] at which half-maximal stimulation of secretion occurred. Quin 2 was used to measure the free [Ca2+] of intact and permeable cells; PRL secretion at a free [Ca2+] corresponding to resting cytoplasmic [Ca2+] was 10% of maximal, while secretion at the [Ca2+] corresponding to the Ca2+ spike induced by thyrotropin-releasing hormone was approximately 25% of maximal.     

Histological study of follicular atresia in the ovary of the domestic hen (Gallus domesticus)

Semi-serial (1 in 20) sections of ovaries were studied and only two types of atresia were identified--non-bursting and bursting. Smaller, non-yolky follicles (less than 1 mm diameter) showed non-bursting atresia. Atresia in follicles greater than 1 mm diameter was invariably of the bursting type which involved the rupture of the follicular wall, and the extrusion of yolk and cellular debris through the rupture site into the stroma. However, this rupture site was small and consequently was not visible in every section but it could always be seen when the follicle was followed in semi-serial sections. The mitotic index of granulosa cells in bursting atretic follicles was much lower than that for normal follicles. The most common criteria for distinguishing non-bursting atretic follicles were the extremely shrunken, irregularly shaped oocytes and the separation of the granulosa from the theca. In bursting atretic follicles, reliable indications were the presence in the ooplasm of some cells or cellular debris, and disorganization of the yolk and granulosa tissue. The presence of pycnotic nuclei in the granulosa cells was not a consistent feature of all atretic follicles of the hen.     

The rate of increase not the amplitude of cytosolic Ca2+ regulates the degree of prolactin secretion induced by depolarizing K+ or hyposmolarity in GH4C1 cells

Depolarizing K+ and medium hyposmolarity caused striking rises in both cytosolic free Ca2+ concentration [( Ca2+]i) and prolactin (PRL) secretion in GH4C1 cells, which were completely blocked by removal of medium Ca2+. However, the increase in [Ca2+]i and PRL secretion induced by hyposmolarity was clearly slower than that induced by K+. Although there was a good correlation between the zenith of PRL secretion and [Ca2+]i induced by various intensities of K+ or hyposmolarity, the regression slopes were significantly different between the K(+)-and hyposmolarity-induced changes (P less than 0.01). There was a good correlation between the maximum rate of change in PRL secretion and that of the increase in [Ca2+]i when the data from the 2 secretagogues were combined (r = 0.994, P less than 0.001, N = 9). We suggest that the rate of increase in [Ca2+]i may be more important than the amplitude of [Ca2+]i in stimulating PRL secretion.     

Effect of GnRH antagonist-induced prolonged follicular phase on follicular atresia and oocyte developmental competence in vitro in superovulated heifers

A GnRH antagonist (Antarelix) was used to suppress endogenous pulsatile secretion of LH and delay the preovulatory LH surge in superovulated heifers to study the effect of a prolonged follicular phase on both follicle and oocyte quality. Oestrous cycles were synchronized in 12 heifers with progestagen (norgestomet) implants for 10 days. On day 4 (day 0 = day of oestrus), heifers were stimulated with 24 mg pFSH for 4 days and luteolysis was induced at day 6 with PGF2 alpha (2 ml Estrumate). Animals in the control group (n = 4) were killed 24 h after the last FSH injection. At this time, heifers in group A36h (n = 4) and group A60h (n = 4) were treated with 1.6 mg of Antarelix every 12 h for 36 and 60 h, respectively, and then killed. After dissection of ovarian follicles, oocytes were collected for individual in vitro maturation, fertilization and culture; follicular fluid was collected for determination of steroid concentrations, and granulosa cells were smeared, fixed and stained for evaluation of pycnosis rates. Granulosa cell smears showed that 90% of follicles were healthy in the control group. In contrast, 36 and 58% of the follicles in group A36h showed signs of early or advanced atresia, respectively, while 90% of the follicles in group A60h showed signs of late atresia. Intrafollicular concentrations of oestradiol decreased (P < 0.0001) from healthy follicles (799.14 +/- 40.65 ng ml-1) to late atretic follicles (3.96 +/- 0.59 ng ml-1). Progesterone concentrations were higher (P < 0.0001) in healthy follicles compared with atretic follicles, irrespective of degree of atresia. Oestradiol:progesterone ratios decreased (P < 0.0001) from healthy (4.58 +/- 0.25) to late atretic follicles (0.07 +/- 0.009). The intrafollicular concentrations of oestradiol and progesterone were significantly higher (P < 0.0001) in the control than in the treated groups. The oestradiol:progesterone ratio was higher (P < 0.0001) in the control (4.55 +/- 0.25) than in the A36h (0.40 +/- 0.05) and A60h (0.07 +/- 0.009) groups. Unexpectedly, the cleavage rate of fertilized oocytes, blastocyst rate and number of cells per blastocyst were not significantly different among control (85%, 41% and 95 +/- 8), A36h (86%, 56% and 93 +/- 5) and A60h (88%, 58% and 79 +/- 4) groups. In addition, there were no significant differences in the blastocyst rates from oocytes derived from healthy (45%), early atretic (54%), advanced atretic (57%) and late atretic follicles (53%). In conclusion, the maintenance of the preovulatory follicles in superovulated heifers with a GnRH antagonist induced more atresia and a decrease in oestradiol and progesterone concentrations. However, the developmental potential in vitro to day 8 of the oocytes recovered from these atretic follicles was not affected.     

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.     

Relationship between progesterone and estradiol contents of follicular fluid and the morphological appearance of granulosa cells of follicles coexisting with corpora lutea in bovine ovaries

Bovine ovaries (n=149) bearing follicles (>5 mm) coexisting with mature corpora lutea (CL;>10 mm) were obtained at a local abattoir without regard for the reproductive status of the donor cows. Most corpora lutea were 21 to 25 mm in diameter, and nearly half of the largest follicles were 11 to 15 mm in diameter. When oocytes were aspirated from follicles 16 to 30 mm in diameter, approximately 60% of them proved to be degenerated. Concentrations of progesterone (P4) and estradiol-17beta (E2) in the follicular fluid of 23 follicles (>10 mm) were determined. Progesterone and estradiol-17beta were found to be the major hormone in 16 (69.6%) and 7 (30.4%) of the follicles, respectively. Light-microscope observations of the granulosa cells of the same 23 follicles showed that 7 were deficient in mural granulosa cells, and that 15 of the remaining 16 follicles were atretic or luteinizing. Ultrastructural observations of granulosa cells revealed many lipid droplets in the cytoplasm of follicles coexisting with mature CL, suggesting the initiation of luteinization. These results show that approximately 70% of the follicles were P4-dominant and that more than 95% of them were morphologically degenerated. Thus it is suggested that morphological signs of atresia precede changes in the concentrations of hormones in the follicular fluid of follicles coexisting with corpora lutea (>10 mm) during the middle of the estrous cycle.     

Source of prolactin in human follicular fluid.

To analyze whether prolactin (PRL) in human follicular fluid (FF) is synthesized locally or derived from the circulation, PRL concentrations of plasma and FF were determined in the patients after ovarian stimulations. The amounts of PRL messenger ribonucleic acid (mRNA) in the follicular tissues during different menstrual phases were also determined. The FF PRL concentration was correlated positively with plasma PRL and highest estradiol levels during the stimulatory cycle. No PRL mRNA sequence was detected in the RNAs extracted from follicles at any stage in the menstrual cycle, although beta-actin mRNA was detected in all samples. In a comparison with pituitary RNA, the PRL mRNA concentration in ovarian follicular tissues seemed to be 10,000 times less than that in the pituitary. These results suggest that FF PRL may not be synthesized locally, but derived from the pituitary via the circulation through passive diffusion, and thus regulated by estrogen.     

In vitro development of individually matured bovine oocytes in relation to follicular wall atresia

Morphologically good-quality cumulus oocyte complexes (COCs) can originate from slightly atretic follicles. Biochemical and ultrastructural investigations reveal that a very high percentage of bovine antral follicles express some degree of atresia. The aim of the present study was to determine the developmental competence of good quality COCs in relation to their biochemically estimated follicular wall apoptosis. For experimental design a single oocyte maturation system was established, followed by group culture processing oocytes together according to their level of follicular wall atresia estimated by an ELISA for apoptotic cell death. Single oocyte culture during maturation reduced the developmental capacity of oocytes significantly (P < 0.01), with 5% blastocysts versus 25% after common group culture. Blastocyst formation for single oocyte maturation was found exclusively in oocytes isolated from luteal stage ovaries with low degree of apoptosis. The level of follicular wall apoptosis in luteal stage follicles (0.79 +/- 0.05 units/mg protein, n = 198) was lower than in follicular stage follicles (1.14 +/- 0.05 units/mg protein, n = 208). This was caused by significant higher levels in small (< 3.5 mm diameter) and large (> 5.5 mm diameter) follicles of the latter group. In conclusion, despite reduced developmental capacity after single oocyte maturation, we were able to reveal some functional relationship between oocyte origin and quality. It was shown that morphologically good quality COCs isolated from follicles with higher degree of apoptosis lose their developmental capacity.     

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)     

Involvement of insulin and growth hormone (GH) during follicular development in the bovine ovary

Insulin and growth hormone (GH) play critical roles in the process of follicular development and maturation. However, the involvement of insulin receptor (IR) and GH receptor (GHR) during follicular development is not well understood. The aim of this study was to investigate the expression of IR and GHR mRNAs in the granulosa cells (GCs) and theca tissues (TCs) of the follicle at different developmental stages (preovulatory dominant follicles, POFs; estrogen-active dominant follicles, EADs; estrogen-inactive dominant follicles, EIDs; and small follicles, SFs), and second, to examine the effects of follicle-stimulating hormone (FSH) and estradiol (E2) on the expression of IR and GHR genes in cultured bovine GCs. Although the concentration of insulin in follicular fluid (FF) was constant at all developmental stages, the GH concentration in FF was significantly increased in the EAD and POF compared with the EID. IR mRNA in GCs and TCs was significantly increased in the POF compared with other follicles. Regarding GHR expression, significant increases of mRNA expression were observed in GCs of EAD compared to those of SF, EID and POF. GHR mRNA in TCs was significantly decreased in the SF compared with other follicles. In cultured GCs, FSH, but not E2, stimulated the expression of IR and GHR genes. Our results suggest that the increase in the expression of GHR may be a turning point for follicles to enter the ovulatory phase during final follicular development and that the insulin system may support the maturation of preovulatory follicles.     

Thyrotropin-releasing hormone-induced spike and plateau in cytosolic free Ca2+ concentrations in pituitary cells. Relation to prolactin release

Using the acetoxymethyl ester of "Quin 2," a fluorescent Ca2+-indicator, we have loaded prolactin (PRL)-producing rat pituitary cells with non-toxic concentrations of Quin 2 and quantitated changes in cytosolic free calcium concentration ( [Ca2+]i) during stimulation of PRL release by thyrotropin-releasing hormone (TRH) and 40 mM K+. TRH induced a biphasic response, with an immediate (less than 1 s) spike in [Ca2+]i from basal levels (350 +/- 80 nM) to a peak of 1-3 microM, which decayed rapidly (t 1/2 = 8 s) to a near basal nadir, then rising to a plateau in [Ca2+]i of 500-800 nM. The TRH-induced spike phase was attenuated but not abolished by prior addition of EGTA, while the plateau phase was eliminated by EGTA. Addition of 40 mM K+ caused an immediate spike in [Ca2+]i to 1-3 microM which equilibrated slowly (t 1/2 = 1 min) directly to a plateau of 600-800 nM. The K+-induced spike and plateau phases were both abolished by prior addition of EGTA. The biphasic nature of TRH action on [Ca2+]i parallels the biphasic actions of TRH on 45Ca2+ fluxes and the biphasic release of PRL by GH cells in suspension. These findings provide evidence that Ca2+-dependent agonist-mediated increases in [Ca2+]i and hormone release are linked, and may generally have two modes: an acute "spike" mode, dependent primarily on redistribution of intracellular Ca2+ stores; and a sustained "plateau" mode, dependent on influx of extracellular Ca2+.     

Expression profiles of key candidate genes involved in steroidogenesis during follicular atresia in the pig ovary

More than 99?% of follicles in mammalian ovaries undergo a degenerative process known as atresia, and thus only a limited number of ovarian follicles actually ovulate after full growth and development. The endocrinological regulatory mechanisms involved in follicular development have been studied extensively, but the precise and systematic molecular mechanisms of steroidogenesis enzymes involved in atresia are unclear. In the present study, we examined whether and how the steroidogenesis enzymes are involved in porcine ovary follicular atresia. Expression of steroidogenic acute regulatory protein, CYP11, CYP17, 3β-hydroxysteroid dehydrogenase (3β-HSD), CYP19, as well as related pituitary and ovarian hormone receptors were quantified in ovaries. During porcine follicular atresia, expressions of P450 cholesterol side chain cleavage enzyme, progesterone and androgen receptors increased significantly during the late atretic stage, while the expression of aromatase and follicle-stimulating hormone receptors decreased significantly in the early stage. These data suggested that the regulation of aromatase by follicle-stimulating hormone might induce follicular atresia, and that progesterone and androgen production further promoted follicular atresia. Additionally, a correlation analysis indicated a large and complex interactive network among these genes and the endocrinological microenvironment of the follicles. Significant correlations were observed between expression of steroidogenic enzymes and their receptors, and also between progesterone and 17β-estradiol (E2) levels in follicular fluid. Taken together, these results suggest that CYP19 plays a role during early atresia by regulating the production of E2, whereas CYP11 and 3β-HSD increase atresia progression by increasing progesterone levels.     

Relationship between concentrations of cortisol in ovarian follicular fluid and various biochemical markers of follicular differentiation in cyclic and anovulatory cattle

Concentrations of cortisol were determined in pooled fluid of small (less than 10 mm) and large (greater than or equal to 10 mm) follicles of cyclic cattle (Exp. 1), and in fluid of the largest follicle of 17 post-partum anovulatory cows (Exp. 2). In Exp. 1, concentrations of cortisol in small follicles were greater (P less than 0.05) than in large follicles (14.7 versus 13.2 ng/ml), and varied significantly with stages of the cycle; small and large follicles had the highest cortisol concentration during the early luteal phase of the cycle. Large follicles had 2-fold greater concentrations of oestradiol than did small follicles, whereas small follicles had 2-fold greater concentrations of androstenedione than did large follicles. Across pools of follicular fluid, cortisol concentrations were correlated only to androstenedione concentrations (r = 0.65, P = 0.07). In Exp. 2, concentrations of cortisol did not significantly differ between oestrogen-active (oestradiol greater than progesterone in follicular fluid) and oestrogen-inactive (progesterone greater than oestradiol) follicles, although oestrogen-active follicles had a 24-fold greater concentration of oestradiol than did oestrogen-inactive follicles. Cortisol concentrations were correlated to hCG binding capacity of thecal cells (r = -0.35, P = 0.08) and to follicular diameter (r = 0.45, P less than 0.05). These results suggest that normally fluctuating concentrations of cortisol in follicular fluid of cattle play little or no active role in follicular differentiation in vivo.