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.     

Levels of messenger ribonucleic acid for cholesterol side-chain cleavage cytochrome P-450 and 3 beta-hydroxysteroid dehydrogenase in bovine preovulatory follicles decrease after the luteinizing hormone surge

During the follicular/luteal phase shift in steroidogenesis, follicular steroid production changes from predominantly estradiol and androgen secretion before the LH surge to decreased androgen and estrogen and increased progesterone after the LH surge. Our objective was to determine whether changes in progesterone production by the preovulatory follicle are effected via changes in mRNA levels for the steroidogenic enzymes cholesterol side-chain cleavage cytochrome P450 (P450scc) and 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD). Bovine preovulatory follicles were obtained in the early follicular phase (n = 9 follicles), the midfollicular phase (n = 4), or the late follicular phase (after the LH surge, but before ovulation; n = 5). Total RNA extracted from granulosa cells and theca interna at the time of cell isolation or after 24 or 72 h of culture in control or LH-containing medium was subjected to Northern analysis, and autoradiographs were scanned densitometrically. P450scc mRNA levels in granulosa cells were high in the early follicular phase and decreased by 96% after the LH surge (P < 0.05). 3 beta HSD mRNA levels in granulosa cells were 4.2-fold higher in early vs. late follicular phase (P < 0.01). In theca interna, 3 beta HSD mRNA levels were 3.6- and 2.6-fold higher in the early vs. the mid- and late follicular phase (P < 0.05), but levels of P450scc mRNA did not differ significantly with stage of follicular development. After granulosa cells had been cultured for 24 h in control or LH-containing medium, P450scc and 3 beta HSD mRNA had declined dramatically compared to mRNA levels at the time of cell isolation during the early follicular phase (P < 0.01). However, after 72 h in control or LH-containing medium, an increase in P450scc and 3 beta HSD mRNA was observed relative to levels at 24 h (P < 0.01). After 72 h of culture, the signal for P450scc and 3 beta HSD mRNA in granulosa cells exposed to LH was higher than the signal detected in cultures without LH (P < 0.01). Similar changes in message for P450scc were observed in cultured thecal cells. Thus, the previously observed increases in production of progesterone by bovine theca interna and granulosa cells obtained after vs. before the LH surge cannot be explained by an increase in message for P450scc and 3 beta HSD.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Apoptosis occurs in granulosa cells but not cumulus cells in the atretic antral follicles in pig ovaries

The porcine antral follicles, 3-6 mm in diameter, were dissected from the ovaries of mature pigs, and then granulosa and cumulus cells were isolated from each follicle. In atretic follicles, high activity of neutral Ca2+/Mg2+-dependent endonuclease and DNA ladder formation, estimated by electrophoresis, were noted in granulosa cells but not in cumulus cells. Extremely low activity of the endonuclease and no DNA ladder formation were observed in both types of cells obtained from healthy follicles. Moreover, apoptotic cells were observed histochemically among granulosa cells only. A good correlation (r = 0.987) between the endonuclease activity of granulosa cells and the progesterone/estradiol ratio of follicular fluid in each follicle was found. These results suggest that apoptosis occurs in granulosa cells but not cumulus cells in the atretic antral follicles in pigs.     

Expression of the steroidogenic acute regulatory protein and luteinizing hormone receptor and their regulation by tumor necrosis factor alpha in rat corpora lutea

Expression of both mRNA and protein of the steroidogenic acute regulatory protein (StAR), in correlation with progesterone (P) production and LH receptor (LHR) mRNA expression, was studied in the corpora lutea (CL) of gonadotropin-induced-pseudopregnant and pregnant rats at various stages of CL development. Immature female rats, 21-22 days old, were injected s.c. with 20 IU eCG to stimulate follicle growth and then with 20 IU hCG 48 h later to induce ovulation. The ovaries were removed at various stages of CL development; either CL were isolated and snap frozen for total RNA analysis, or whole ovaries were fixed in Bouin's fluid for paraffin sectioning. The results of in situ hybridization, immunohistochemistry, and Northern blotting showed that the increase in StAR mRNA and protein expression was well correlated with the increase in serum P concentration. StAR expression was restricted to the luteal cells or theca cells in antral follicles. Both StAR mRNA and protein in the CL of pseudopregnant rats increased steadily on Day 1 and Day 4, reached highest levels on Day 4, and then dropped sharply on Day 8 when luteolysis takes place. LHR mRNA content was high on Day 1 but dropped significantly on Day 2. LHR mRNA increased to high levels on Day 4 and 8 and then declined on Day 12. StAR mRNA and protein levels in the CL of pregnant rats were high during early luteal development (Day 2, 4), increased even further on Day 9, and decreased on Day 13 when luteolysis takes place. It is therefore suggested that the expression of StAR coincides well with the capacity of P production in the CL and that StAR expression can be used as a functional "marker" of CL development. To study the possible effect of cytokines on StAR expression, pseudopregnant rats on Day 5 were injected s.c. with 10 IU hCG plus 20 microg prolactin (PRL), with or without 500 IU tumor necrosis factor alpha (TNFalpha) 30 min later. TNFalpha significantly inhibited hCG/PRL-induced StAR and LHR mRNA expression at 1 and 3 h post-TNFalpha. It is suggested that the luteolytic effect of TNFalpha may be mediated by its direct inhibition on StAR expression or by an indirect decrease in LHR expression.     

Treatment with progesterone and 17 beta-oestradiol to induce emergence of a newly-recruited dominant ovulatory follicle during oestrus synchronisation with long-term use of norgestomet in Brahman heifers

The aim of this study was to determine the effect on ovarian follicular growth and atresia, of acute treatment with either 100 mg of progesterone (n = 10), 200 mg of progesterone (n = 10), 10 mg of oestradiol + 100 mg of progesterone (n = 10), 10 mg of oestradiol (n = 10) or no treatment (n = 10), given on Day 10 of a 17-day treatment with a norgestomet implant in randomly cycling Bos indicus heifers. The fate of the dominant follicle on Day 10, emergence of the new cohort of follicles and the intervals from implant removal to ovulation were recorded by ultrasonography. Plasma concentrations of Luteinizing hormone (LH), progesterone and oestradiol were determined during the time when the norgestomet implant was in place. All treatments resulted in the emergence of a new cohort of follicles within 5 days of administration. The day of emergence of the ovulatory follicle tended to be delayed after treatment with 100 mg of progesterone (2.7 +/- 0.3 days after treatment), 200 mg of progesterone (3.7 +/- 0.5 days after treatment), 10 mg of oestradiol + 100 mg of progesterone (4.4 +/- 0.2 days after treatment) and 10 mg of oestradiol (4.6 +/- 0.4 days after treatment) compared to control heifers (1.4 +/- 1.4 days after time of treatment). The mean interval from implant removal to onset of oestrus was significantly shorter after treatment with 100 mg of progesterone (38.4 +/- 2.6 h) than after treatment with 200 mg of progesterone (61.5 +/- 3.9 h) but otherwise, the mean interval from implant removal to onset of oestrus did not differ. Oestrus synchrony, measured by the sample standard deviation of oestrus onset, was tighter in all treatment groups compared to untreated control heifers. The mean interval from implant removal to ovulation did not differ significantly between groups. The synchrony of ovulation, measured by the sample standard deviation of the interval from implant removal to ovulation, was significantly tighter after treatment with 100 mg of progesterone, 200 mg of progesterone and 10 mg of oestradiol compared to control heifers. Treatment with 10 mg of oestradiol resulted in ovulation in seven of 10 heifers before implant removal, three of which failed to ovulate after implant removal. Progesterone administered on Day 10 lowered plasma LH concentrations (P < 0.05), whereas treatment with oestradiol caused a surge of LH and ovulation. Progesterone administered with oestradiol prevented the LH surge. A combination treatment of oestradiol and progesterone given on Day 10 of a 17-day norgestomet treatment in a range of follicular states resulted in the consistent emergence of a new cohort of follicles which included the eventual ovulatory follicle.     

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.     

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)     

Differential responses of granulosa cells from small and large follicles to follicle stimulating hormone (FSH) during the menstrual cycle and acyclicity: effects of tumour necrosis factor-alpha

This study determined effects of follicle stimulating hormone (FSH) alone and in combination with tumour necrosis factor (TNF), on granulosa cells from small (5-10 mm diameter) and large (>10-25 mm) follicles during follicular and luteal phases of the cycle and during periods of acyclicity. Granulosa cells were collected from ovaries of premenopausal women undergoing oophorectomy. The cells were cultured with human FSH (2 ng/ml) and testosterone (1 microM) in the presence or absence of human TNF-alpha (20 ng/ml). Media were removed at 48 and 96 h after culture and progesterone, oestradiol and cAMP in media were measured by radioimmunoassays. FSH stimulated the accumulation of oestradiol from granulosa cells of small follicles during the follicular and luteal phases but not during acyclicity; and TNF reduced oestradiol accumulation in the presence of FSH. Interestingly, in granulosa cells from small follicles, progesterone and cAMP secretion increased in response to FSH and neither was affected by TNF. Thus, TNF specifically inhibited the conversion of testosterone to oestradiol in granulosa cells from small follicles. FSH stimulated oestradiol production by granulosa cells of large follicles obtained only during the follicular phase of the cycle and TNF inhibited the FSH-induced oestradiol secretion. Granulosa cells obtained from large follicles during the luteal phase and during acyclicity did not accumulate oestradiol in response to FSH. However, FSH increased progesterone and cAMP secretion by granulosa cells obtained from large follicles during the follicular and luteal phases. During the luteal phase alone, TNF in combination with FSH increased progesterone accumulation above that of FSH alone. FSH did not increase progesterone, oestradiol or cAMP secretion by granulosa cells obtained from large follicles during acyclicity. Thus, FSH increases progesterone, oestradiol and cAMP secretion by granulosa cells of small follicles during the follicular and luteal phases and TNF appears to inhibit FSH-induced oestradiol secretion specifically in those cells. In large follicles, FSH-stimulated granulosa cell secretion of oestradiol is limited to the follicular phase and this effect can be inhibited by TNF. In addition, when granulosa cells of large follicles do not increase oestradiol secretion in response to FSH, TNF stimulates progesterone secretion.     

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.     

Premature response to luteinizing hormone of granulosa cells from anovulatory women with polycystic ovary syndrome: relevance to mechanism of anovulation

Polycystic ovary syndrome is the most common cause of anovulatory infertility. Anovulation in polycystic ovary syndrome is characterized by the failure of selection of a dominant follicle with arrest of follicle development at the 5-10 mm stage. In an attempt to elucidate the mechanism of anovulation associated with this disorder we have investigated at what follicle size human granulosa cells from normal and polycystic ovaries respond to LH. Granulosa cells were isolated from individual follicles from unstimulated human ovaries and cultured in vitro in serum-free medium 199 in the presence of LH or FSH. At the end of a 48-h incubation period, estradiol (E2) and progesterone (P) were determined in the granulosa cell-conditioned medium by RIA. In ovulatory subjects (with either normal ovaries or polycystic ovaries), granulosa cells responded to LH once follicles reached 9.5/10 mm. In contrast, granulosa cells from anovulatory women with polycystic ovaries responded to LH in smaller follicles of 4 mm. Granulosa cells from anovulatory women with polycystic ovaries were significantly more responsive to LH than granulosa cells from ovulatory women with normal ovaries or polycystic ovaries (E2, P < 0.0003; P, P < 0.03). The median (and range) fold increase in estradiol and progesterone production in response to LH in granulosa cell cultures from size-matched follicles 8 mm or smaller were E2, 1.0 (0.5-3.9) and P, 1.0 (0.3-2.5) in ovulatory women and E2, 1.4 (0.7-25.4) and P, 1.3 (0.3-7.0) in anovulatory women. Granulosa cells from anovulatory (but not ovulatory) women with polycystic ovaries prematurely respond to LH; this may be important in the mechanism of anovulation in this common endocrinopathy.     

Modulation of ovarian cytochrome P450 17 alpha-hydroxylase and cytochrome aromatase messenger ribonucleic acid by prolactin in the domestic turkey

The effect of exogenous ovine prolactin (oPRL) on preovulatory follicle P450 17 alpha-hydroxylase (C17) and aromatase (ARO) mRNA abundance was investigated in turkeys. Ovine PRL (124 IU/hen per day) was injected i.m. into four sets (n = 8) of laying turkeys for 2, 4, 8, or 14 days. Vehicle was injected into control hens for 8 days (n = 8). Blood samples were collected and serum was assayed for LH, progesterone (P), testosterone (T), and estradiol (E). Theca layers from the largest (F1) and the third (F3), fifth (F5), and seventh (F7) largest preovulatory follicles and from small white follicles (SWF) were examined for C17 and ARO mRNA contents. The number of atretic follicles increased from 0 (vehicle-injected controls) to 9 (14-day-oPRL-injected hens). Serum E, T, and LH levels decreased, while P levels remained unchanged. There was a transient increase in theca C17 mRNA abundance of 2- and 4-day-oPRL-treated hen follicles. Cytochrome P450 ARO mRNA levels were reduced in SWF and F7 in response to oPRL. Thecal C17 and ARO mRNA content was reduced during follicular maturation in laying hens. ARO mRNA was not detectable in granulosa cells. The progressive decline in C17 and ARO mRNA content associated with follicular maturation as well as the absence of ARO mRNA in granulosa cells is consistent with the secretory activity of P, T, and E in preovulatory follicles. These findings suggest that reduced circulating E may be a consequence of suppressed ARO gene expression whereas the oPRL suppression of T secretion may not be coupled to C17 gene expression.     

Selection, dominance and atresia of follicles during the oestrous cycle of heifers

This study examined the correlation between measurement of follicle growth by ultrasound, and measurement of intrafollicular ratios of oestradiol and progesterone concentrations and the serum concentrations of FSH during selection, dominance and atresia or ovulation of dominant follicles in heifers. Heifers were ovariectomized on days 0 (before LH surge), 1 (after LH surge, preovulation), 1 (postovulation), 3, 6 and 12 of the oestrous cycle. Blood samples were collected at 4-6 h intervals. After ovariectomy all follicles > or = 5 mm were measured and follicular fluid was aspirated. Follicles were classified by size according to ultrasound (F1, largest; F2, second largest; F3, all remaining follicles > or = 5 mm) and by the ratio of oestradiol:progesterone concentrations. During the follicular phase, a single dominant oestrogen-active follicle increased in diameter while serum concentrations of LH increased and FSH decreased (P < 0.05). On day 1 (after LH surge, preovulation), serum LH and FSH decreased to pre-surge concentrations (P < 0.0001), while follicle size and intrafollicular progesterone concentration increased and oestradiol concentration decreased (P < 0.05). A dominant nonovulatory follicle, classified as oestrogen-active on days 1, 3 and 6 and oestrogen-inactive on day 12, increased in size from day 1 to day 7 and lost dominance during days 10-12, coincident with the growth of multiple oestrogen-active follicles. The serum FSH concentration increased transiently (P < 0.05) before each new wave of dominant follicular growth. The overall correlation of ultrasound measurements of follicle diameter with measures of follicle size after ovariectomy was high.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Developmental changes in marmoset granulosa cell responsiveness to insulin-like growth factor-I: interactions with follicle-stimulating hormone and estradiol

The biological actions of insulin-like growth factor-I (IGF-I) on granulosa cell steroidogenesis at defined stages of preovulatory follicular development in the marmoset monkey were examined. Studies were carried out by primary cell culture of granulosa cells derived from small antral (0.5-1.mm diameter) and large preovulatory (2-3.mm diameter) follicles collected during the mid-late follicular phase of the ovarian cycle. IGF-I (0.3-100 ng/ml) had no effect on progesterone accumulation or aromatase activity during 48-h culture of granulosa cells from small follicles. Progesterone accumulation by cells from large follicles was also unaffected by IGF-I over the same time period, although aromatase activity was stimulated in a dose-dependent manner (18-fold increase over basal levels with a maximally stimulatory dose of 30 ng IGF-I/ml). In contrast, granulosa cells from small and large follicles responded to IGF-I in terms of both progesterone accumulation and aromatase activity after longer periods of culture (4 days for progesterone; 6 days for aromatase). Concurrent treatment of granulosa cells from small follicles with estradiol (10(-7) M) enhanced the dose-dependent actions of IGF-I on both indices of steroidogenesis and advanced the time at which IGF-I stimulated activity was first detectable. The effects of estradiol on granulosa cell IGF-I responsiveness were independent of cell number. A synergistic action of IGF-I on FSH-stimulated granulosa cell steroidogenesis was not apparent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in ovarian function and gonadotropin secretion preceding the onset of nutritionally induced anestrus in Bos indicus heifers

The aim of this study was to monitor endocrine and ovarian changes immediately preceding the onset of nutritionally induced anestrus. Daily blood samples were obtained from 14 postpubertal heifers for one estrous cycle (initial estrous cycle). Subsequently, heifers designated "restricted" were given a limited diet (n = 9), and daily blood samples were obtained for approximately 21 days preceding onset of anestrus (anovulatory cycle). Controls were allowed ad libitum dietary intake (n = 5), and daily blood samples were collected for a complete estrous cycle during a time period that coincided with that preceding onset of anestrus in restricted heifers. Plasma samples were assayed for LH, FSH, progesterone, and estradiol-17 beta. The ovaries of all heifers were examined daily using transrectal ultrasonography from the initial until the final or anovulatory estrous cycles to determine changes in growth of follicles and corpora lutea. Anestrus was defined as failure of ovulation of the dominant follicle following luteolysis. When anovulatory and initial estrous cycles in restricted heifers were compared, mean concentrations of LH were lower (p < 0.01), and diameters of dominant follicles were smaller (p < 0.01); mean concentrations of estradiol-17 beta were also lower in the three days following luteolysis (p = 0.06), but concentrations of FSH appeared to be higher (p = 0.003); maximum diameters of corpora lutea were smaller (p < 0.001), but duration of luteal phases and concentrations of progesterone preceding luteolysis were similar (p > 0.10). In controls, no differences were found between estrous cycles for any of these variables. It is concluded that failure of ovulation, following reduced dietary intake, resulted from insufficient circulating LH to stimulate maturation of the ovulatory follicle.     

Growth and cellular proliferation of antral follicles throughout the follicular phase of the estrous cycle in Meishan gilts

Meishan gilts were ovariectomized 2 h after an i.v. injection of 5'-bromo-2'-deoxyuridine (BrdU, a thymidine analogue; 5 mg/kg body weight) on Days 15-19 of the estrous cycle or 24-30 h after observed estrus (post LH, PLH). All antral follicles > or = 3 mm from one ovary were fixed in Carnoy's solution. Granulosa and thecal cell labeling indexes (LI; percentage of nuclei staining for BrdU) as well as LI of cells within the basal, middle, and antral thirds of the granulosa cell layer were estimated for each follicle. In addition, antral and granulosa cell layer volume, granulosa cell layer thickness, granulosa cell density, number of granulosa cells, and number of S-phase cells per hour were estimated for each follicle. Mean follicular diameter increased linearly (p < 0.01) from Day 15 to PLH, with a growth rate of 0.77 mm/day. Granulosa and thecal cell LI decreased (p < 0.01) from Day 15 to PLH; however, granulosa cell LI was greater (p < 0.01) than thecal cell LI on Days 15 and 16 but less (p < 0.05) than thecal cell LI on Day 19. Follicles collected from PLH gilts contained no labeled granulosa cells. Cells within the basal third of the granulosa cell layer contained fewer (p < 0.01) labeled nuclei than did cells within the middle or antral thirds. In addition, LI within the basal and middle thirds of the granulosa cell layer decreased (p < 0.01) from Days 15 to 18 and from Days 15 to 17, respectively, whereas LI within the antral third remained constant from Days 15 to 18. Granulosa cell layer thickness was greatest (p < 0.01) on Day 15, then decreased (p < 0.01) and was similar from Day 16 to PLH. Granulosa cell density was similar from Days 15 to 19, then decreased (p < 0.01) for PLH gilts. Antral and granulosa cell layer volumes increased linearly (p < 0.01) from Days 15 to 19 and Day 15 to PLH, respectively, resulting in 2.8 and 1.9 volume doublings and doubling times of 1.4 and 2.7 days, respectively. Number of granulosa cells per follicle increased linearly (p < 0.01) from Day 15 to PLH, resulting in 1.5 cell doublings and a doubling time of 3.3 days. Number of S-phase cells per follicle per hour was similar from Days 15 to 18 and then decreased (p > 0.01) from Day 18 to PLH. In summary, the percentages of proliferating granulosa and thecal cells decreased throughout the final stages of antral follicular development. Differentiation of granulosa cells occurred from the basal to the antral area as follicles matured. We proposed that, during the latter stages of follicular development, the rapid increase in follicular diameter resulted primarily from expansion of the antral cavity, whereas increases in the granulosa cell layer volume and number of granulosa cells per follicle maintained a constant granulosa cell layer thickness.