Onset Of Luteal Regression Research Articles

Quantification of Immune Cells in the Primate Corpus Luteum During the Menstrual Cycle; Increases During the Late Luteal Phase Indicate a Potential Role in Luteolysis.

The potential role of the immune system in regulation of the mammalian ovary has primarily been investigated in domestic animal and rodent models; however the presence of immune cells in the human ovary has also been noted by immunohistochemistry. Recently, microarray-identified luteal mRNAs for immune regulatory components such as chemokines and cytokines, immune cell-associated genes, as well as gene signaling pathways associated with immune function were found to fluctuate during the lifespan of rhesus macaque corpora lutea (CL). To characterize immune cell populations in primate CL throughout the menstrual cycle, luteal tissue was collected from rhesus females at discrete intervals of the luteal phase, including early days 3-5 (n=3), mid days 6-8 (n=4), mid-late day 10 (n=4) and day 12 (n=3), as well as after onset of luteal regression (2 days n=2, and 3-4 days n=4, following a drop in serum progesterone levels to < 0.3 ng/ml). Luteal cells were enzymatically dispersed, counted and viability assessed. Wet weight (mg) of CL were significantly different across the luteal phase (P<0.03), but total numbers of viable cells did not differ between groups (P=0.8). Dispersed cells were then incubated with fluorescently-labeled antibodies specific for immune-cell surface proteins: CD11b (ITGAM; neutrophils including activated lymphocytes, granulocytes and monocytes), CD14 (monocytes/macrophages), CD16 (FCGR3; primarily natural killer cells as well as neutrophils and macrophages), and CD3 (CD3-epsilon/T cell receptor complex; T lymphocytes). Number of labeled cells for each CL (positive events in 10,000 cells counted) was assessed using a BD LSR II flow cytometry instrument for each antibody (FCS Express version 3, De Novo Software). A non-immune, isotype antibody matched for each cell surface specific marker was used to gate the positive cells within the CL. The number of positive events recorded for immune cells at each stage of the luteal phase was then analyzed by one-way ANOVA (SAS version 2.0). Both CD11b- and CD14-positive cells (neutrophils and macrophages) were constant until increasing significantly 3-4 days after onset of luteal regression (P<0.0001). Numbers of CD16-positive (natural killer cells) increased initially between early and mid luteal phase to plateau through the mid-late stages, until a further 3-fold increase by 3-4 days after onset of regression (P<0.005). Additionally, CD16-positive cells were the most abundant immune cell type in CL during the mid and mid-late luteal phases, and increased to levels comparable with CD11b-positive cells during luteal regression. Presence of CD3-positive lymphocytes tended to increase 3-4 days after regression (P=0.07), but were less abundant than other cell types. These data demonstrate a remarkable increase in cells of the innate immune system, (i.e. macrophages and neutrophils) in the primate CL associated with the latter stages of luteal regression. Natural killer cells may have a dual role in the CL to promote luteal structure and function during the mid-luteal phase, whereas macrophages, neutrophils and natural killer cells may participate in structural luetolysis at the end of the luteal lifespan. Supported by NIH grant R01HD020869 (RLS) and NCRR grant P51RR000163 (Support For National Primate Research Center).

Ovarian structures during the estrous cycle and early pregnancy in ewes.

The development of ovarian follicular and luteal structures during the estrous cycle and early pregnancy in ewes was examined. Ewes were treated with prostaglandin F2 alpha to induce estrus; they were placed with either two fertile or two vasectomized rams and observed for estrus twice daily. Beginning at estrus (Day 0), ovaries were scanned for CL and for follicles > or = 2 mm by transrectal ultrasonography daily for the first 25 days of pregnancy (PREG, n = 17 ewes) or until Day 8 of the second estrous cycle (CYC, n = 15 ewes). Jugular blood samples were collected at ultrasonography and on Days 30, 35, and 40 of gestation for RIA of progesterone and estradiol-17 beta. Total follicles (17.6 +/- 1.3), number of follicles > or = 4 mm in diameter (11.3 +/- 0.6), and number of follicles that became the largest during the first 16 days of pregnancy or the first estrous cycle (4.9 +/- 0.3) did not differ between PREG and CYC ewes. Number of newly detected follicles each day and difference in mean diameter between the largest two follicles and all other follicles were similar between PREG and CYC ewes until luteal regression. At that time, difference in diameter between the largest two follicles and all other follicles increased in CYC ewes in parallel with estradiol-17 beta in serum. Mean concentrations of progesterone in serum were higher in PREG than in CYC ewes (2.4 +/- 0.1 vs. 2.0 +/- 0.1 ng/ml; p < 0.05) before onset of luteal regression, but mean areas of CL did not differ.(ABSTRACT TRUNCATED AT 250 WORDS)

Uterine Secretion of Prostaglandin F2α in Response to Oxytocin in Ewes: Changes during the Estrous Cycle and Early Pregnancy1

Experiment 1 was conducted to determine when the ovine uterus develops the ability to secrete prostaglandin F2 alpha (PGF2 alpha) in response to oxytocin and how development is affected by pregnancy. Pregnant and nonpregnant ewes received an injection of oxytocin (10 IU, i.v.) on Day 10, 13, or 16 postestrus. Jugular venous blood samples were collected for 2 h after injection for quantification of 13,14-dihydro-15-keto-PGF2 alpha (PGFM). In nonpregnant ewes, concentrations of PGFM increased following oxytocin on Day 16 but not on Day 10 or 13. Concentrations of PGFM did not increase following treatment on Day 10, 13, or 16 in pregnant ewes. Therefore, the ability of oxytocin to induce uterine secretion of PGF2 alpha develops after Day 13 in nonpregnant but not in pregnant ewes. Experiment 2 was conducted to precisely define when uterine secretory responsiveness to oxytocin develops. Pregnant and nonpregnant ewes received oxytocin on Day 12, 13, 14, or 15. In nonpregnant ewes, concentrations of PGFM increased following treatment on Days 14 and 15, but not earlier. Peripheral concentrations of progesterone showed that uterine secretory responsiveness to oxytocin developed prior to the onset of luteal regression. As in experiment 1, the increase in concentrations of PGFM following administration of oxytocin was much lower in pregnant than in nonpregnant ewes; however, some pregnant ewes did respond to oxytocin with an increase in PGFM. In experiment 3, pregnant ewes received an injection of oxytocin on Day 18, 24, or 30 postmating. Concentrations of PGFM increased following oxytocin on Days 18 and 24. The conceptus appears to delay and attenuate the development of uterine secretory responsiveness to oxytocin.

Factors affecting the origin of the ovulatory follicle in heifers with induced luteolysis

Seventeen nulliparous Holstein heifers were treated with a luteolytic dose of prostaglandin F 2α (PGF) on Day 8 (Day 0 = day of pretreatment ovulation) when the dominant follicle of the first follicular wave (Wave 1) was expected to be in the static phase. Wave 2 was first detected on Days 8 or 9 in 2 of the 14 and 3 of the 3 heifers that ovulated from Waves 1 and 2, respectively. That is, most heifers treated with PGF on Day 8 ovulated from the dominant follicle of Wave 1, except when Wave 2 emerged early, in heifers that ovulated from Wave 1, the dominant follicle increased ( P < 0.0001) in diameter from Day 8 to the day prior to post-treatment ovulation (mean increase, 2.3 mm), even though it had apparently reached its maximum diameter before the induction of luteolysis. Heifers ovulating from the dominant follicle of Wave 1 differed from heifers ovulating from the dominant follicle of Wave 2 in the following ways: change in diameter of the ovulatory follicle from day of treatment to day of post-treatment ovulation (means, 2.3 versus 9.0 mm, P < 0.0001), diameter of Wave-1 dominant follicle on the day prior to post-treatment ovulation (means, 17.6 versus 13.7 mm, P < 0.0005); and length of the interval from day of treatment to day of post-treatment ovulation (means, 4.2 versus 6.3 days, P < 0.03). Following ovulation after the PGF-induced luteolysis, 11 heifers had two-wave estrous cycles and 5 had three-wave cycles. Three-wave cycles were associated with an earlier emergence of Wave 2 and a longer luteal phase (onset of luteal regression; Day 20.0 versus Day 17.5; P < 0.03).

Effects of Recombinant Bovine Somatotropin on Luteinizing Hormone and Ovarian Function in Lactating Dairy Cows1

Thirty-two lactating Holstein cows were assigned to 1 of 4 groups in a randomized block design using a 2 X 2 factorial arrangement of treatments. Recombinant bovine growth hormone (rbSt; 25 mg/day) or placebo was administered beginning at Day 35 or 70 postpartum. All cows began treatment approximately 3 days post-estrus. Blood samples were collected at least once daily for a 70-day period to determine the concentration of progesterone and the duration of the luteal and follicular phases. During estrous cycles 1 and 3, frequent blood samples were taken (every 10 min for 8 h) 24 and 60 h after the onset of luteal regression. These samples were assayed for luteinizing hormone (LH), and samples coincident with the second LH pulse detected were assayed for estradiol. Ultrasonography was used to determine the size of the largest ovarian follicle from Day 17 until ovulation in estrous cycles 1 and 3. Luteal life span, length of the follicular phase, and diameter of the largest follicle were not affected by treatment with rbSt. Administration of rbSt increased the concentration of progesterone in plasma during the first two luteal phases (p less than 0.01). Progesterone was elevated during the mid-luteal phase of cycle 3 in rbSt-treated cows that began treatment about Day 35 postpartum but not in cows that began treatment on Day 70 postpartum (Treatment X Stage X Day, p less than 0.01). During the first follicular phase studied, LH pulse frequency was higher (p = 0.06) in rbSt-treated cows than in cows receiving the placebo.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Reduced Luteinizing Hormone Concentrations on Corpus Luteum Function during the Menstrual Cycle of Rhesus Monkeys*

To further define the relationship between plasma LH concentrations and progesterone secretion by the primate corpus luteum, we examined luteal function in rhesus monkeys in response to reduced LH concentrations during the luteal phase of the menstrual cycle. Five anovulatory rhesus monkeys received a pulsatile infusion of synthetic GnRH (6 micrograms/pulse; one pulse per h, iv) to restore menstrual cyclicity. During the early luteal phase (4-5 days after ovulation), the amount of GnRH administered per pulse was reduced to 1/250th or 1/750th of the standard GnRH infusion regimen. Plasma LH concentrations, determined by bioassay, were reduced by approximately 50% during cycles maintained by reduced GnRH concentrations compared with the standard GnRH dosage. Serum progesterone concentrations were maintained for 5-6 days after GnRH reduction and declined thereafter, and premature menstruations were observed in four of seven cycles maintained by the 1/250th GnRH reduction and four of six cycles maintained with the 1/750th GnRH reduction. These results are consistent with the hypothesis that luteal regression during the nonfertile menstrual cycles of primates is due primarily to an alteration in luteal cell responsiveness to LH, rather than a reduction in the gonadotropic drive to the corpus luteum per se. When plasma LH concentrations were reduced during the early luteal phase to values below those found during the onset of luteal regression in control cycles, luteal function was maintained for 5-6 days. However, as the luteal phase progressed, the reduced LH concentrations were unable to sustain progesterone secretion, and premature menses occurred in some, but not all, animals.

Chronic treatment with bromocryptine during the oestrous cycle of the cow: Evidence that prolactin is not involved in preovulatory follicular growth

Nine heifers received an intramuscular injection of 15 mg of bromocryptine twice daily from Day 1 (the day of first standing oestrus defined as Day 0) until 50 h after the onset of luteal regression. At this time the ovaries were collected and the number and quality of follicles > 2 mm were determined. Binding of 125I-labelled hCG and 125I-labelled ovine prolactin to follicles > 5 mm and concentrations of progesterone, oestradiol and androstenedione in the follicular fluid were estimated. Plasma concentrations of prolactin, LH and oestradiol were studied from the onset of luteal regression until ovariectomy. Seven heifers served as a control group. Prolactin was effectively suppressed during bromocryptine treatment (0.21 ± 0.01 μg/ l, mean ± SEM; n = 9) as compared to control heifers (16.8 ± 3.6 μg/ l; n = 7). Concentrations of LH were not affected by the low level of prolactin (0.99 ± 0.08 μg/ l vs. 0.95 ± 0.11 μg/1). Both groups showed a similar time-course of the oestradiol concentration in the peripheral blood during the 50-h period. No specific binding of prolactin to granulosa cells and thecal tissue of follicles > 5 mm could be demonstrated, regardless of their quality or diameter. Preovulatory follicles of bromocryptine-treated and control heifers did not differ with regard to the number of specific binding sites for hCG and the concentrations of oestradiol, progesterone and androstenedione in the follicular fluid. It is concluded that prolactin is not involved in the growth and development of antral follicles during the normal oestrous cycle of the cow.

Effects of different gonadotropin pulse frequencies on corpus luteum function during the menstrual cycle of rhesus monkeys.

In the nonfertile menstrual cycle, the frequency of episodic LH secretion declines from approximately 1 pulse/h in the early luteal phase to 1 pulse/4-8 h in the mid- to late luteal phase, but the relevance of this phenomenon to the initiation of functional luteal regression is not completely understood. We investigated whether a reduction in LH pulse frequency causes a decline in luteal progesterone production by experimentally reducing LH pulse frequency during the early luteal phase, and measured the effects on the subsequent plasma progesterone pattern and the onset of luteal regression. Rhesus monkeys were rendered anovulatory by placing radiofrequency lesions in the arcuate region of the medial basal hypothalamus or surgically transecting the hypothalamic-pituitary stalk. Endogenous gonadotropin secretion and ovulatory menstrual cycles were restored by pulsatile infusion of synthetic GnRH at a frequency of 1 pulse/h. Commencing on days 3-6 of the luteal phase, GnRH frequency was changed to either 1 pulse/8 h (four animals) or 1 pulse/24 h (four animals), or maintained at the standard 1 pulse/h frequency (four animals). Luteal phases of 13- to 17-day duration were observed in all animals kept on the 1 pulse/h frequency and in three of four animals in which the frequency was changed to 1 pulse/8 h on day 3 of the luteal phase. Daily midluteal phase (days 5-10) plasma progesterone levels observed in response to the 1 pulse/h and 1 pulse/8 h infusion regimens were similar (mean +/- SE, 4.1 +/- 0.4 vs. 3.2 +/- 0.4 ng/ml; P greater than 0.1). In contrast, short luteal phases were observed in all animals after the LH pulse frequency was reduced to 1 pulse/24 h. Comparison of plasma LH responses to a representative GnRH pulse of each GnRH infusion regimen revealed that the maximal LH levels attained in response to 1 pulse/8 h (47.5 +/- 11.5 ng/ml) were significantly greater (P less than 0.05) than the maximal LH levels attained in response to 1 pulse/h (30.5 +/- 3.2 ng/ml) or 1 pulse/24 h (27.2 +/- 5.0 ng/ml). Progesterone levels remained elevated for 140-200 min after the LH pulse resulting from the 1 pulse/8 h infusion regimen. In response to the 1 pulse/24 h infusion regimen, plasma progesterone levels remained elevated for 60 min after the LH pulse.(ABSTRACT TRUNCATED AT 400 WORDS)

Morphometry of the functional and regressing corpus luteum of the guinea pig

A morphometric study of functional and regressing corpora lutea (CL) of guinea pigs (n = 5 per day) was performed on days 9, 12, and 16 of the estrous cycle. On day 9 the functional CL contained congruent to 750,000 cells, which included 565,200 +/- 56,700 (S.D.) endothelial cells or pericytes and 137,300 +/- 7,700 luteal cells. Between days 9 and 12 the only significant change suggesting the onset of regression was a reduction in vascular luminal surface area. During this time the number of luteal cells per CL increased to 204,400 +/- 34,800 (P less than .05), with an accompanying reduction in luteal cell volume from 19.8 +/- 1.8 to 14.4 +/- 2.4 pl/cell (P less than .01). The increase in cell numbers was explicable by cell division, with mitotic indices of 0.83% and 0.97% on days 9 and 12, respectively. Luteal volume was unaltered. Between days 12 and 16, the mean volume of a single CL fell from 3.98 +/- 0.2 to 1.42 +/- 0.3 mm3 (P less than .01) and luteal cell volume was reduced to 5.3 +/- 1.1 pl/cell (P less than .01). Between these 2 days the number of endothelial cells per CL fell from 539,900 +/- 75,500 to 144,400 +/- 63,300 (P less than .01), with an accompanying reduction in vascular luminal surface area and in the volume occupied by vascular lumina. The total number of luteal cells per CL was not reduced significantly. It was concluded that luteal cell numbers in the guinea pig increase up to the time of onset of luteal regression, and that during regression up to day 16, shrinkage of luteal cells is the major cause of loss of luteal volume. During regression, endothelial cell loss occurs much more rapidly than loss of luteal cells.

Adenylate Cyclase Activity of Porcine Corpora Lutea1, 2

Adenylate cyclase of porcine luteal tissue has been studied in relation to luteal regression. The level of adenylate cyclase activity was greater in tissue obtained at Days 12 or 14 of a cycle than in tissue from Days 16 or 17. The enzyme was responsive to added LH at Days 12 or 14 of the estrous cycle, but failed to respond to LH in tissue obtained at Days 16 or 17 of a cycle. A decline in adenylate cyclase activity and loss of responsiveness to LH appear to be correlated with the onset of luteal regression.