Follicular Fluid Estradiol-17 Beta Research Articles

Follicular development and maturation in gilts selected for an index of high ovulation rate and high prenatal survival1

Seventy-one 10th-generation gilts from White Line-1 (WL-1 = randomly selected control line) and White Line-2 (WL-2 = selected for an index of ovulation rate and prenatal survival rate) were used to compare the pattern of follicular development and atresia during the follicular phase of the estrous cycle. Gilts were treated with PGF(2alpha)on d 13 of the estrous cycle (d 0 of induced follicular development) to induce luteolysis and assigned randomly within line and sire for ovary recovery on d 0, 2, 3, 4, 5, and the day after estrus. Ovaries were evaluated for numbers of corpora albicantia and small (2 to 2.9 mm), medium (M1 = 3 to 4.9 mm; M2 = 5 to 6.9 mm), and large (>or=7 mm) follicles. The concentration of estradiol-17beta in follicular fluid was used to classify individual M2 and large follicles as estrogen-active (>or=100 ng of estradiol-17beta/mL) or inactive (<100 ng of estradiol-17beta/mL). The WL-2 gilts had a greater ovulation rate than WL-1 gilts at their pre-treatment estrus (20.4 vs. 13.8 corpora albicantia; P < 0.001). The small and M1 follicle populations decreased rapidly in both lines over time (P < 0.001). The M2 follicle population increased in both lines between d 0 to 4 and then decreased. Mean estradiol concentration of M2 follicles increased in both genetic lines over time (P < 0.02). All large follicles were estrogen-active in both lines; the number of large follicles increased with day (P < 0.001) and was similar in both lines. The number of estrogen-active M2 follicles was similar in both lines, increasing to d 3 and 4 and then decreasing (P < 0.01) thereafter. However, the total number of estrogen-active follicles (sum of estrogen-active M2 and large follicles) was greater in WL-2 than in WL-1 gilts (P < 0.04), increasing to the ovulatory potential by d 3 in WL-1 gilts, but continuing to increase through d 4 in WL-2 gilts. Selection of an additional six ovulatory follicles from the estrogen-active M2 follicle pool after d 5 was required in both lines to achieve the projected ovulation rate, and after estrus, the number of large follicles remained insufficient to attain the ovulatory potential of each line.

Follicular atresia in pigs: measurement and physiology.

The physiological regulation of follicular atresia was investigated during the early luteal phase after ovulation and during altrenogest-synchronized preovulatory maturation in pigs (gilts). Apoptosis in dispersed granulosa cells was determined by flow cytometry. Apoptotic (A0) cells contain low, subdiploid amounts of DNA fluorescence. Follicles were classified biochemically as atretic or nonatretic based on the percentage of A0 (% A0) cells, atretic with > or = 10%, and nonatretic with < 10% A0 granulosa cells. The % A0 granulosa cells/follicle ranged from .02 to 89. Follicles containing debris in their isolated granulosa cells were classified as morphologically atretic. The morphological and biochemical criteria of atresia were in agreement for 224 of 248 follicles. Internucleosomal DNA cleavage, the hallmark of apoptosis, was determined by autoradiographic analysis of [32P]3'-end labeled DNA from granulosa cells. Densitometric analysis showed that optical density of [32P]3'-end labeled DNA fragments in the .18 to 20 kbp size range was correlated with the % A0 cells (R > .9, n = 22, P < .001). During altrenogest-synchronized preovulatory maturation, < 5% of large (> 6 mm in diameter) follicles were atretic. Among medium-sized follicles (3 to 6 mm) on d 1 and 3 of preovulatory maturation, only 17% were atretic, in contrast with d 5 when 87% were atretic. During the early luteal phase, atretic follicles/pig increased from 6% on d 5 to 50% on d 7 after estrus. Follicular fluid estradiol-17 beta concentration was greater (P < .001) in nonatretic than in atretic follicles on d 5 and 6 after estrus, but by d 7 estradiol-17 beta had decreased to a mean < 1 ng/mL in nonatretic and atretic follicles. The increase in apoptosis in granulosa cells and loss of estradiol-17 beta production in vivo indicated a high incidence of atresia among the first group of follicles grown after ovulation in pigs. These results indicate that apoptotic cell death was involved in degeneration of granulosa cells and atresia during two different stages of follicular development.

Steroidogenesis in the Preovulatory Porcine Follicle1

The expression of four major steroidogenic enzymes in porcine theca and granulosa cell layers of preovulatory follicles was related to the levels of steroids in follicular fluid and gonadotropin concentrations in peripheral serum at slaughter. Ovaries were collected during proestrus, early estrus, and late estrus as evidenced by behavioral signs. Follicles were dissected from the ovaries, and theca, granulosa, and follicular fluids were pooled for each of 24 sows. Cytochromes P450 17 alpha-hydroxylase/17-20 lyase (P450c17), aromatase (P450arom) and side-chain cleavage (P450scc), as well as 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), were subjected to Northern and Western immunoblot analyses. The concentrations of estradiol-17 beta, testosterone, androstenedione, and progesterone were determined in follicular fluid, and peripheral serum was assayed for estradiol-17 beta, LH, and FSH. Stages of preovulatory development were verified by plasma levels of LH, FSH, and estradiol-17 beta. Thecca expressed P450c17, P450arom, P450scc, and 3 beta HSD whereas granulosa expressed only P450arom and low levels of P450scc. Thecal P450c17, thecal P450arom, and granulosa P450arom expression decreased coincidentally as serum estradiol-17 beta and follicular fluid estradiol-17 beta, testosterone, and androstenedione levels declined after the presumed gonadotropin surge. Unlike P450c17 and P450arom P450scc and 3 beta HSD remained relatively constant in theca and granulosa. From these data, we suggest that the theca interna may be the primary steroidogenic compartment of the porcine follicle during its final stages of preovulatory development. Moreover, preovulatory estrogen secretion appears to be controlled by the coordinated expression of a triad of enzymes in the porcine follicle that includes theca P450c17, theca P450arom and granulosa P450arom.

Steroidogenic cytochrome P450 enzyme messenger ribonucleic acids and follicular fluid steroids in individual follicles during preovulatory maturation in the pig.

Changes in follicular concentrations of steroidogenic cytochrome P450 enzyme mRNAs were determined during preovulatory maturation. RNA was isolated from 59 individual follicles dissected from 18 pigs during altrenogest-synchronized preovulatory follicular maturation: at Day 1 (pre-follicular phase), Day 3 (early follicular phase), Day 5 (mid-follicular phase), and Day 7 (late follicular phase, 24-36 h after the onset of the LH surge). Follicular fluid was aspirated for steroid RIA. RNA was also isolated from pooled granulosa cells, theca tissue, and luteal tissue. RNA was analyzed by Northern and slot-blot procedures using cDNA probes to human aromatase cytochrome P450 (P450arom), porcine 17 alpha-hydroxylase cytochrome P450 (P450(17) alpha), and porcine cholesterol side-chain cleavage cytochrome P450 (P450scc). P450arom mRNA was expressed in both granulosa and theca interna cells but was not detectable in luteal tissue from Day 13 of the estrous cycle. Follicular P450arom mRNA concentration tended to increase between Days 1 and 5 and decreased (p < or = 0.05) by 92% between Days 5 and 7. Follicular fluid estradiol-17 beta concentration increased 17-fold between Days 1 and 5 and then decreased (p < or = 0.05) by 96% between Days 5 and 7. P450(17) alpha mRNA was present in theca interna but was not detected in granulosa cells or luteal tissue. Follicular P450(17) alpha mRNA concentrations did not differ significantly among days, but the content per follicle increased (p < 0.05) between Days 1 and 5 and decreased (p < 0.05) between Days 5 and 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of a bovine growth hormone transgene inhibits pregnant mare's serum gonadotropin-induced follicle maturation in prepuberal gilts.

Prepuberal gilts were injected with PMSG to determine whether expression of a bovine growth hormone (bGH) transgene inhibited preovulatory maturation of ovarian follicles. Seven transgenic (TG) gilts of line 3706, which expresses a mouse metallothionein-bGH transgene, and eight nontransgenic, control (C) gilts (128 to 147 d old) were injected with PMSG, 12.5 IU/kg BW, 72 h before necropsy. Surface ovarian follicles > or = 1 mm in diameter were counted, measured for diameter, and aspirated for fluid. Follicles were classified morphologically as healthy or atretic and those with follicular fluid estradiol-17 beta > or = 100 ng/mL were classified as estrogenactive (EA). The number of follicles per gilt was 64.3 +/- 6.1 (mean +/- SEM) and did not differ significantly between bGH-TG and C gilts. The PMSG treatment induced growth of large (> 5 mm) follicles in both bGH-TG and C gilts. However, compared with C gilts, bGH-TG gilts had fewer (P < .05) large follicles (5.9 +/- 1.5 vs 18.3 +/- 5.4), a lower proportion of EA large follicles (35 +/- 12.5 vs 69 +/- 13.2%), and in large follicles less (P < .05) estradiol-17 beta (86 +/- 17 vs 350 +/- 69 ng/mL) and androstenedione (300 +/- 33 vs 1,283 +/- 221 ng/mL). Follicular fluid progesterone and inhibin did not differ significantly between bGH-TG and C gilts. The incidence of atresia among small and medium follicles did not differ significantly between bGH-TG and C gilts.(ABSTRACT TRUNCATED AT 250 WORDS)

Innate variability in sexual development irrespective of body fatness in gilts1

To separate the effects of fatness from those of lean tissue growth on reproductive development, 52 gilts (33.2 kg, 75 d) had ad libitum access to a high-energy diet (HE; n = 31) or were fed restrictively a low-energy diet (approximately 80% of ad libitum; LE; n = 31) until 160 d of age. All HE and 15 LE gilts were then slaughtered; the 16 LE gilts that remained were then allowed ad libitum access to the low-energy diet until slaughter at 175 d (LER). Twelve littermate trios allocated across treatments were cannulated and bled during both unfed and fed states before slaughter. Gilts in the HE group were 8 kg heavier, 3.5 mm fatter (backfat), and consumed 14.9 MJ of DE more per day than gilts in the LE group at 160 d (hereafter P < .05); LER gilts were 7 kg heavier, 2.2 mm fatter, and consumed 21 MJ of DE more per day at 175 d than LE gilts at 160 d. Carcass fat was lower in LE gilts than in HE or LER gilts, but carcass lean was similar among groups. Treatment did not affect basal or episodic LH or growth hormone secretion, reproductive tract weight, follicular volume, or plasma or follicular fluid estradiol-17 beta. Postprandial plasma insulin was greater in LER than in HE or LE gilts, but plasma insulin-like growth factor I and urea nitrogen were similar among groups. Plasma nonesterified fatty acids and creatinine were greater in HE than in LE or LER gilts. These results suggest that when protein accretion rate is maximal, differences in fatness do not influence reproductive development in the prepubertal gilt. Irrespective of treatment, the only measured factor showing an association with ovarian development was the innate variability in episodic and basal LH secretion.

Effect of Estrogen Administration on Endogenous and Luteinizing Hormone-Releasing-Hormone-Induced Luteinizing Hormone Secretion and Follicular Development in the Lactating Sow1

The objectives of this study were to investigate whether estradiol treatment during lactation modifies 1) the patterns of endogenous LH, FSH, and prolactin (PRL) release; 2) the sensitivity of the pituitary to exogenous injections of LHRH; and 3) the responsiveness of the ovarian follicles to gonadotropin. Plasma LH, FSH, and PRL were determined in samples taken repeatedly from 18 sows on Days 24-27 of lactation. Ovaries were then recovered, and follicular development was assessed by measuring the follicular diameter (FFD) and follicular fluid estradiol-17 beta concentration (FFE) of the ten largest follicles dissected from each ovary. Sows were randomly allocated to one of four treatments: 1) Group C (4 sows) received no treatment; 2) Group LHRH (5 sows) received 800 ng of LHRH every 2 h throughout the sampling period; 3) Group E2 (4 sows) received subcutaneous implants containing estradiol-17 beta 24 h after start of sampling; 4) Group LHRH + E2 (5 sows) were administered a combination of LHRH and estradiol-17 beta implants. Between-animal variability for plasma LH, FSH, and PRL was considerable. LH concentration and LH pulse frequency increased (p less than 0.05) after LHRH treatment in the LHRH and LHRH + E2 groups; however, an acute inhibition of LH secretion was observed in the latter group immediately after estradiol implant application. In the absence of LHRH treatment, estradiol caused chronic inhibition of LH secretion. Follicular development was greater in the LHRH and LHRH + E2 groups compared to the C and E2 groups (p less than 0.05 for both FFD and FFE).(ABSTRACT TRUNCATED AT 250 WORDS)

Growth and differentiation of large antral follicles after spontaneous luteolysis in heifers: changes in concentration of hormones in follicular fluid and specific binding of gonadotropins to follicles.

The objective of the present study was to characterize changes in growth, concentrations of steroids in follicular fluid (FF) and gonadotropin receptors of individual antral follicles after spontaneous luteolysis in heifers and to reexamine our hormonal method of classifying healthy and atretic follicles. Groups of heifers were ovariectomized (OVX) before regression of corpora lutea on d 17, 18 and 19 of an estrous cycle and after regression of corpora lutea at time periods before, during and after preovulatory gonadotropin surges. Each follicle greater than or equal to 6 mm in diameter was examined for concentration of progesterone, androstenedione, testosterone and estradiol-17 beta in FF and capacities of granulosal and thecal cells to specifically bind 125I-bovine follicle stimulating hormone (bFSH) or 125I-human chorionic gonadotropin (hCG). Follicles were separated into two classes, estrogen-active (E-A) or estrogen-inactive (E-I). Concentrations of estradiol in FF were higher than both progesterone and androgens in E-A follicles, whereas either androgens or progesterone was higher than estradiol in E-I follicles. Diameters, volumes of FF and numbers of granulosal cells in E-A follicles increased from d 17 through the end of the preovulatory gonadotropin surges. During this time, concentrations of progesterone, androstenedione and estradiol in FF and specific binding of 125I-hCG to granulosal and thecal cells increased, whereas specific binding of 125I-bFSH to granulosal cells decreased. In E-I follicles, progesterone in FF decreased and androstenedione increased from d 17 through end of gonadotropin surges. The E-I follicles had a higher incidence of granulosal cells with pycnosis than E-A follicles. We concluded that changes in growth, concentration of steroids in FF and gonadotropin receptors in E-A follicles after spontaneous luteolysis were similar to changes we previously reported for E-A follicles after prostaglandin-induced luteolysis, and that ratio of concentration of estradiol in FF to both progesterone and androgens could be used to separate potential ovulatory (E-A) from atretic (E-I) follicles in heifers.

Ovarian function in the cycling cow: relationship between gonadotropin binding to theca and granulosa and steroidogenesis in individual follicles.

Thirty-six Angus and Angus crossbred cows were used in a 3 x 2 x 2 factorial experiment designed to determine the effects of stage of the estrous cycle (day 6, 12 or 18), a long term with a single ovary (2 years) and side of previous ovulation or remaining ovary on the development of large ovarian follicles (LF; diameter greater than or equal to 8 mm). Follicular fluid estradiol-17 beta (FFE) concentration, localization of gonadotropin binding sites and granulosa cell condition were determined for each LF by radioimmunoassay, autoradiography and histological examination, respectively. No side differences were noted. However, the sample was biased by the use of equal numbers of left and right side ovulators. Compared to intact controls, one-ovary cows showed 100% compensation of LF development. Eighty-five percent of the LF studied bound follicle stimulating hormone (FSH) in the granulosa and human chorionic gonadotropin (hCG) in the theca. Thirty-eight percent also bound hCG in the granulosa. Both binding patterns were found on all days studied. Atretic follicles had lower (P less than .05) FFE concentrations than healthy follicles (r = -.59). The number of LF per cow increased (P less than .05) from day 6 (1.3) to days 12 (1.8) and 18 (2.1). Conversely, their FFE content per cow decreased (P less than .05) from 109 ng/cow on day 6 to 20 ng on; day 12 and 34 ng on day 18. The data suggested the emergency of two LF classes on day 18, one preovulatory, with exceptionally high FFE levels, the other atretic or becoming atretic, with low FFE levels.