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Biomedical subjects

A M Dharmarajan

Publications and source records attributed to A M Dharmarajan.

At least 55 records · Page 3Linked to original sources
PubMed1991

Changes in ultrastructure of transport systems and in rates of progestin secretion in the corpus luteum during late pregnancy in the rat.

Morphometric studies have confirmed that the corpus luteum (CL) of the pregnant rat contains luteal cells with numerous microvilli which directly face an extensive network of sinusoidal capillaries. From this it has been suggested that extensive development of transport structures is necessary to support progesterone synthesis and secretion. The present study was carried out to determine whether these transport structures could be related quantitatively to different rates of total progestin (progesterone plus 20 alpha-hydroxypregn-4-en-3-one) secretion reported to be 32, 10, and 23 micrograms/hr per ovary on day 16 and the morning (AM) and afternoon (PM) of day 22, respectively. Histological analysis was carried out on two CL, fixed by immersion, from each of five rats, at each stage of gestation. The important findings to emerge were that when the progestin secretion rate was greater, there was a significant increase in surface specializations on the luteal cell and a thickening of the capillary walls. There was also a greater volume of interstitial space between luteal cells and capillaries. However, due to the development of microvilli and unevenness in the capillary wall, the physiological diffusion distance (harmonic distance) between luteal cell cytoplasm and blood was not increased. Collectively, these results show that changes in the rate of progestin secretion are accompanied by significant, although disproportionate, changes in transport structures and suggest that the latter are important in supporting luteal function.

Animals↗
PubMed1991

Do prostaglandins lead to ovulation in the rabbit by stimulating proteolytic enzyme activity?

OBJECTIVE: To determine if prostaglandins (PGs) have a direct effect on the ovarian proteolytic enzyme system by examining ultrastructure of the follicle wall and the microvasculature in the presence and absence of indomethacin and by using the isolated perfused rabbit ovary. DESIGN: Nine hours after administration of human chorionic gonadotropin (hCG) or hCG plus indomethacin, follicles were removed and processed for scanning and transmission electron microscopy. Isolated perfused rabbit ovaries were induced to ovulate with PGF2 alpha (100 ng/mL) in the presence and absence of tranexamic acid (0.1, 1.0, or 10 mM), a plasminogen activator inhibitor. RESULTS: The addition of indomethacin to hCG inhibited ovulation and production of PGs without affecting the follicular microvasculature. However, the changes in follicle wall architecture were less pronounced after treatment with indomethacin. Ovulatory efficiency in response to PGF2 alpha (the percent of follicles greater than 1.5 mm that ovulate) was significantly reduced (P less than 0.01) by 10 mM tranexamic acid. CONCLUSIONS: These results suggest that PGs induce follicular rupture by activation of proteolytic enzymes located in the follicle wall.

Animals↗
PubMed1991

Estradiol regulation of the rabbit corpus luteum: in vivo and in vitro studies.

The objective of this study was to determine whether estradiol has a direct effect on progesterone secretion by the rabbit corpus luteum. Empty or estradiol-filled Silastic capsules were implanted sc into pseudopregnant rabbits (day 0). Ten days later (day 10), peripheral blood was obtained via the marginal ear vein, and Silastic capsules were removed. Twenty-four hours after capsule removal (day 11), blood samples were obtained and ovaries removed for in vitro perfusion. The artery and vein of each ovary were individually cannulated, and ovaries were perfused in vitro for 6 h. Mean progesterone secretion rates were determined from perfusate samples taken every 30 min. On day 10, serum progesterone concentrations were similar in control and estradiol-treated animals. On day 11, 24 h after withdrawal of Silastic capsules, serum progesterone concentration in the estradiol-treated rabbits decreased significantly compared to controls. The withdrawal of estradiol also significantly reduced the secretion of progesterone by in vitro perfused ovaries in estradiol-withdrawn rabbits compared to empty capsule controls. Addition of estradiol or 25-hydroxycholesterol (25-OH) to the perfusion medium significantly increased progesterone secretion by ovaries from estradiol-withdrawn rabbits but not to control values. In contrast, a combination of estradiol plus 25-OH restored progesterone secretion to control levels. Although estradiol together with 25-OH stimulated progesterone secretion 24 h after estradiol withdrawal, progesterone secretion in vitro was unaffected 48 h after capsule removal, whereas pregnenolone stimulated secretion 5-fold. These results demonstrate that estradiol has a direct and acute stimulatory effect on progesterone secretion by the rabbit corpus luteum.

Animals↗
PubMed1991

Effect of inhibition of oxygen free radical on ovulation and progesterone production by the in-vitro perfused rabbit ovary.

The potential role of oxygen free radicals in hCG-induced ovulation was investigated using the free radical scavenging enzymes superoxide dismutase (SOD) and/or catalase with the in-vitro perfused rabbit ovary preparation. SOD (25 micrograms/ml) and SOD + catalase (25 micrograms/ml) significantly reduced the % of large follicles that ovulated during perfusion (P less than 0.005). Neither maturity nor degeneration of ovulated ova and follicular oocytes was affected by SOD and/or catalase. Progesterone concentration in the perfusate was significantly increased in the SOD + catalase treatment group (P less than 0.01). These results indicate a significant role for oxygen free radicals in the process of ovulation.

Animals↗
PubMed1991

Direct effect of angiotensin II on in-vitro perfused rabbit ovary.

The effects of angiotensin II (AII) and its receptor blocker, saralasin (SAR), on ovulation and oocyte maturation were investigated in an isolated, in-vitro perfused rabbit ovary. Ovulation and oocyte maturation were induced by AII in the absence of human chorionic gonadotrophin (hCG). SAR inhibited ovulation induced by AII or hCG, but not oocyte maturation. AII appears to play a critical role in follicle rupture, but not in resumption of oocyte meiosis.

Angiotensin II↗
PubMed1990

Transforming growth factor-beta 1 inhibits ovarian androgen production: gene expression, cellular localization, mechanisms(s), and site(s) of action.

It is the aim of this study to establish ovarian transforming growth factor-beta 1 (TGF beta 1) gene expression, to reevaluate its cellular localization, and to explore potential interactions of this regulatory peptide on ovarian androgen biosynthesis. Northern analysis of whole ovarian polyadenylated RNA revealed a single 2.5-kilobase transcript corresponding to the TGF beta 1 precursor. Immunohistochemical staining localized the protein to the thecal-interstitial (interfollicular) compartment. To explore potential autocrine effects of TGF beta 1, use was made of whole ovarian dispersates from immature rats the differentiation of which was monitored by the acquisition of androgen biosynthetic capacity. The accumulation of androsterone, the major androgenic steroid detectable in this culture system, increased 5.4-fold over baseline in response to treatment with hCG (1 ng/ml). This effect was further optimized (2- to 4-fold) by supplementation with insulin (1 microgram/ml) and insulin-like growth factor-I (50 ng/ml). In the absence of these optimizing supplements, TGF beta 1 (10 ng/ml) was without effect on basal androsterone accumulation, producing distinct, albeit relatively limited (25%), inhibition of hCG hormonal action. In contrast, supplement-mediated optimization of ovarian androgen biosynthesis revealed TGF beta 1 to be a highly potent inhibitor (greater than 80%) of hCG hormonal action. This reversible TGF beta 1 action proved time and dose dependent, with a minimal time requirement of 72 h and a median inhibitory dose of 2.6 ng/ml. TGF beta 1 action was not due to diminution in the viable cell mass or altered cAMP generation and, therefore, most likely involved a site(s) of action distal to or independent of cAMP generation. Cellular radiolabeling studies of TGF beta 1-treated ovarian cells disclosed the accumulation of steroid intermediates proximal to the 17 alpha-hydroxylation step, suggesting TGF beta 1-mediated blockade at the level of the steroidogenic enzyme 17 alpha-hydroxylase/17-20-lyase. Taken together, these observations are in keeping with the view that TGF beta 1, possibly of thecal-interstitial origin, may not only play a positive paracrine role at the level of the adjacent granulosa cell (as previously reported), but may also constitute one of several autocrine signals concerned with the regulation of ovarian androgen economy. As such, these findings reaffirm the polyfunctional nature of TGF beta 1 action, as manifested by its diametrically opposed effects in different ovarian compartments.

Androgens↗
PubMed1989

Effects of systemic administration of indomethacin on ovulation, luteinization, and steroidogenesis in the rabbit ovary.

Indomethacin blocks ovulation in human chorionic gonadotropin-stimulated rabbits. Experiments were done with an in vitro ovarian perfusion system to investigate whether indomethacin affects luteinization and steroidogenesis. Indomethacin (10 mg/kg) was administered in combination with human chorionic gonadotropin (100 IU) via a marginal ear vein, and a second dose of indomethacin was given 8 hours later. Control animals received vehicle in place of indomethacin. Laparotomy was performed 24 hours after the initial treatment. The presence of unruptured follicles and corpora lutea was recorded and the ovaries were perfused in vitro for 3 hours. Progesterone, prostaglandin F2 alpha, prostaglandin E2, and 6-keto-prostaglandin F1 alpha were measured in samples obtained at 0, 30, 60, 120, and 180 minutes from the circulating perfusion medium entering and exiting the ovary. At the end of the perfusion all ovaries (12 treated and 10 controls) were fixed for histologic analysis. Ovulation occurred in all control ovaries but in none of the indomethacin-treated ovaries. The mean number of unruptured follicles per ovary in the treated group was not significantly different from the number of corpora lutea plus unruptured follicles per ovary in the controls. Cells in both groups were qualitatively similar in ultrastructure; abundant lipid droplets, smooth endoplasmic reticulum, and mitochondria were seen. Secretion rates of progesterone and prostaglandin did not differ between the two groups during the 3-hour perfusion period. These results suggest that transformation of granulosa cells into fully functional luteal cells can occur in the absence of follicular rupture.

Animals↗
PubMed1989

In vivo administration of allopurinol affects ovulation and early embryonic development in rabbits.

The potential role of the hypoxanthine-xanthine oxidase system in human chorionic gonadotropin-induced ovulation, nuclear maturation, and preimplantation development was investigated in the rabbit by use of the xanthine oxidase inhibitor allopurinol. Allopurinol (50 mg/kg) or vehicle was injected, followed by human chorionic gonadotropin (100 IU). Administration of inhibitor or vehicle was repeated 3 hours later. Ovaries and ovulated ova were inspected 12 or 24 hours after human chorionic gonadotropin administration for follicle rupture, cumulus dispersal, and nuclear maturation. Ova were inseminated in vitro and assessed for development to the blastocyst stage, up to 96 hours after insemination. Allopurinol significantly reduced ovulatory efficiency, defined as the percent of large follicles that ovulate, and decreased the percent of ova with cumulus dispersal. Allopurinol significantly inhibited morula and blastocyst formation. These data suggest that the xanthine oxidase system may play a role in ovulation and early embryonic development.

Allopurinol↗
PubMed1989

Changes in ovarian blood flow and secretion of progesterone and 20 alpha-hydroxypregn-4-en-3-one on day 16 and the morning and afternoon of day 22 of pregnancy in the rat.

During late pregnancy in rats, ovarian secretion of progesterone decreases and that of its reduced metabolite, 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OHP), increases. The present study was undertaken to determine whether changes in ovarian blood flow are consistent with changes in progestin secretion. Rats (n = 5 per group) were examined on Day 16, the time of maximal progesterone secretion, and in the morning (AM) and afternoon (PM) of Day 22, the day prior to parturition. Ovarian blood flow was monitored continuously for 60 to 80 min, and serial samples of arterial and ovarian venous blood were obtained at 20-min intervals for determination of ovarian secretion rates of progesterone and 20 alpha-OHP. Ovarian blood flow increased from 0.38 +/- 0.04 ml/min (mean +/- SEM) on Day 16, to 0.77 +/- 0.05 and 0.78 +/- 0.04 ml/min on Day 22 AM and PM, respectively, whereas the secretion of progesterone decreased from 26.9 +/- 4.0 to 4.5 +/- 1.0 and 3.2 +/- 0.3 micrograms/h per ovary. The secretion of 20 alpha-OHP was similar on Day 16 and Day 22 AM (5.6 +/- 1.7 and 5.4 +/- 1.3 micrograms/h per ovary) but then increased to 18.9 +/- 1.2 micrograms/h per ovary by Day 22 PM. Thus the amount of total progestins secreted per unit rate of blood flow relative to that on Day 16 (100%) fell to 15% and 34% on the morning and afternoon of Day 22, respectively. Clearly, the relative changes in ovarian progestin secretion and blood flow in the rat near term to not conform to patterns observed at luteal regression in some other species.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Prostaglandins and progesterone secretion in the in vitro perfused pseudopregnant rabbit ovary.

The relationship between progesterone and prostaglandin (PG) secretion in the pseudopregnant rabbit corpus luteum was investigated using isolated in vitro perfused ovaries. Progesterone and PG secretion were measured on days 1, 11, and 18 of hCG-induced pseudopregnancy. The mean progesterone secretion increased significantly from days 1 to 11, and then decreased significantly by day 18. PG secretion was inversely correlated with progesterone secretion, suggesting that PG might inhibit progesterone secretion. To test this hypothesis, indomethacin, an inhibitor of PG secretion, was administered to intact rabbits from days 11-18 of pseudopregnancy and/or on day 18 ovaries were perfused in vitro with indomethacin. Indomethacin administered in vivo, in vitro, or both in vivo and in vitro significantly reduced PG secretion compared to that in controls, but did not affect progesterone secretion. In addition, perfusion of ovaries in vitro with PGF2 alpha did not alter progesterone secretion on either day 11 or day 18. Thus, although there is an inverse relationship between progesterone and PG secretion during pseudopregnancy, PGF2 alpha alone had no effect on progesterone secretion. These results question the hypothesis that PGF2 alpha alone is the luteolytic factor.

6-Ketoprostaglandin F1 alpha↗
PubMed1989

In-vivo and in-vitro determination of components of rabbit early pregnancy factors.

Two types of rabbit early pregnancy factor (EPF) components, prepared from the in-vitro perfused ovary and oviduct and from serum ammonium sulphate fractions, were investigated to elucidate the source of EPF production. The state of the animals, i.e. pregnant, pseudopregnant, unstimulated or platelet activating factor (PAF)-treated, and order of addition of the components to the assay lymphocytes were varied to characterize conditions of production and expression of EPF activity. Although each component alone had no EPF activity, combination of two components, i.e. ovary and oviduct components, or serum precipitate and supernatant components, expressed EPF activity. The oviduct and serum supernatant components were produced in pregnancy and pseudopregnancy but the ovary and serum precipitate components were produced only in pregnancy. The similarity of the production pattern and ability of the perfusate and serum components to yield EPF activity when combined suggests that they are similar or the same. The sources of stimulation of EPF production did not appear to affect component production because the activity produced by the perfused ovary and oviduct in pregnancy or in response to PAF stimulation appeared similar. Oviduct and supernatant components apparently bound directly to lymphocytes. These results suggest that EPF components are produced in the ovary and oviduct individually and that the combination of the two components expresses EPF activity in the rabbit.

Animals↗
PubMed1989

Quantitative comparison of luteal histology in the rat and rabbit: changes from mid- to late gestation.

Progesterone secretion by the corpora lutea (CL) of rats and rabbits declines from a peak, at about Day 16 of gestation, to near term (Day 22 rats and 28 rabbits). However there are major differences between the two species in CL growth and blood flow over this period. In the present work quantitative histological measurements were made of CL at these stages to examine the accompanying structural changes. Eight rats and five rabbits were examined at each stage: standard morphometric techniques were used. There was gross discrepancy between the two species in the histology of their CL at peak secretory activity. Although the proportions of the major tissue components were similar, the rabbit luteal cell (52 pl) was five times larger than that in the rat (9 pl). There was substantially less vascular and interstitial space in the rabbit, all characteristics which might affect transport processes between luteal cell cytoplasm and blood. Over the period examined, there was no change in CL volume in the rat but a 37% reduction in the rabbit due to loss of luteal cells. The vascular space in the rat, however, was reduced, whereas that in the rabbit declined only in proportion to the overall decrease in CL volume. These results show that structurally there is a substantial difference in patterns of early regression between the two species which reflect different mechanisms involved.

Animals↗
PubMed1988

Platelet activating factor-induced early pregnancy factor activity from the perfused rabbit ovary and oviduct.

Early pregnancy factor, a glycoprotein, and platelet activating factor, a phospholipid, are early signals of pregnancy appearing during the preimplantation period. The aim of this study was to determine if the administration of platelet activating factor to rabbits would stimulate the production of early pregnancy factor and, if so, to ascertain the organs responsible. Early pregnancy factor was assayed after the administration of synthetic platelet activating factor to intact rabbits, rabbits that had undergone bilateral salpingo-oophorectomy, and isolated in vitro perfused rabbit ovary and/or oviduct. Although serum early pregnancy factor activity was detectable 30 minutes after platelet activating factor injection in intact rabbits, no activity was found in rabbits whose ovaries and oviducts had been removed. Perfusate early pregnancy factor activity was detectable only in the presence of both ovary and oviduct, and was first evident 120 minutes after platelet activating factor exposure. However, combined samples of perfusates from the ovary alone and oviduct alone expressed positive early pregnancy factor activity. Furthermore, the addition of platelet activating factor to either single or combined perfusates did not increase early pregnancy factor activity. Our data imply that platelet activating factor induces the production of ovary and oviduct early pregnancy factor components independently.

Animals↗
PubMed1988

Effects of prostacyclin on ovulation and microvasculature of the in vitro perfused rabbit ovary.

Prostacyclin (prostaglandin I2) methyl ester at 0.1, 1, or 10 micrograms/ml was added to the perfusate of one rabbit ovary every 2 hours for the first 10 hours of perfusion. The contralateral ovary was perfused with medium alone. Prostacyclin methyl ester at 1 and 10 micrograms/ml induced ovulation in the absence of gonadotropin, with ovulatory efficiencies of 26.7% +/- 3.8% and 46.5% +/- 5.0%, respectively. Most ovulated ova (77.4%) did not progress beyond the germinal vesicle stage, and there was no significant degeneration of ovulated ova or follicular oocytes. Examination of the follicular microvasculature 5 hours after exposure to prostacyclin revealed marked vessel dilatation and filling defects at the apex. By 7 hours after prostacyclin exposure, the intrafollicular space contained extravasated resin, reflecting increased vascular permeability. The vascular changes paralleled those previously observed in gonadotropin-induced ovulation. These results indicate that prostacyclin acting locally alters the vascular integrity of the follicle wall and facilitates follicle rupture.

Animals↗
PubMed1988

Quantitative light microscopic analysis of corpus luteum growth during pseudopregnancy in the rabbit.

We employed stereological methods at the light-microscope level to examine the mechanism by which corpora lutea (CL) grow during the course of pseudopregnancy in the rabbit. Corpus luteum volume per ovary, the absolute volume of luteal cells per CL, individual luteal cell volume, the number of luteal and endothelial cells per CL, and capillary surface area per CL were examined in rabbits at Days 1, 4, 7, 11, and 18 of pseudopregnancy. Total CL volume increased from 3.7 +/- 0.1 microliter to 30.3 +/- 0.5 microliter over Days 1 to 11 and thereafter decreased to 15.2 +/- 1.1 microliter by Day 18. Stereological analyses showed that the increases in CL volume from Day 1 to Day 11 were due primarily to increases in the volume of individual luteal cells (from 2.6 +/- 0.2 pl on Day 1 to 23.5 +/- 1.7 pl on Day 11, 1 pl = (10 mu)3; r = 0.96), and that the decrease in CL volume after Day 11 resulted largely from a decrease in luteal cell volume (to 12.8 +/- 1.5 pl). In contrast, no change was seen in the number of luteal cells per CL (range 9.1 x 10(5)-12.5 x 10(5)). These data show that CL growth and subsequent regression during pseudopregnancy result primarily from changes in the volume of individual luteal cells, and not from changes in the number of luteal cells. These data support the hypothesis that modulation of progesterone production during pseudopregnancy is due to changes in individual luteal cell volume and not to changes in cell number.

Animals↗
PubMed1988

Progesterone secretion by corpora lutea of the isolated perfused rabbit ovary during pseudopregnancy.

An ovarian in vitro perfusion method was adapted to examine rabbit corpus luteum (CL) function during pseudopregnancy. Ovaries were perfused in vitro with tissue culture Medium 199 with or without 3% bovine serum albumin (BSA). Samples were obtained from both arterial and venous cannulae, allowing rates of progesterone secretion to be determined. Two perfusion methods were compared: a closed system in which perfusion medium continuously recirculated through the tissue, and an open system in which the ovarian vein cannula was left outside the perfusion chamber. The addition of 3% BSA was found to prevent edema and distortion of the interstitial space, and to result in increased progesterone secretion. With the closed perfusion system, the progesterone secretion rate measured over the 6-h perfusion was significantly higher on Day 11 than on Day 1 of pseudopregnancy and had declined significantly on Day 18. There was no difference in secretion rates whether the perfusion system was open or closed, and the results obtained with both systems closely approximated in vivo progesterone secretion rates. Measurements of CL tissue progesterone content before and after in vitro perfusion indicated that the changes in progesterone secretion seen during pseudopregnancy resulted from differences in the synthesis and secretion of progesterone and not from leakage of progesterone already present in the CL prior to perfusion. Taken together, these results indicate that the modified in vitro perfused rabbit ovary preparation described herein is an appropriate model to examine progesterone secretion by ovaries bearing CL.

Animals↗
PubMed1988

Detection of early pregnancy factor (EPF) using the rabbit ovary and oviduct perfused in vitro.

Two peaks of rabbit serum EPF activity were seen over the course of pregnancy. Rabbit ovaries with or without attached oviducts were perfused in vitro for 5 h beginning 12, 16, 24, 48, 72 and 120 h after mating. Perfused isolated ovaries did not produce EPF in vitro, but significant EPF activity was detected in the perfusate of the ovary together with oviduct. Pseudopregnant animals and those rabbits that did not ovulate exhibited no perfusate EPF activity. Perfusate EPF activity was highest at the time embryos were at the pronuclear stage and continued through the morula stage. Although the location of embryos at 72 h after mating varied between oviduct and uterus, EPF activity was maintained over the perfusion period. The results suggest that EPF release occurs within 3 h of fertilization and that the presence of the preblastocyst embryo is crucial for EPF release.

Animals↗