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Three treatment protocols were used in 156 in vitro fertilization cycles. Leuprolide acetate was begun on day 1 of the cycle in one group (n = 20), on day 3 in another (n = 48), and the third control group (n = 88) did not receive the gonadotropin-releasing hormone analog. Human menopausal gonadotropin was initiated on day 3 in all groups. Peak estradiol (E2) levels and the mean numbers of mature oocytes and embryos transferred per cycle were significantly greater in the day 3 group than in either the day 1 or control groups. Patients who received the day 3 protocol had significantly fewer cancelled cycles. A decline in E2 was observed on the third day of analog administration in certain patients, particularly those on the day 1 protocol. Follicle-stimulating hormone and luteinizing hormone (LH) levels increased two- to fivefold 24 hours after initiation of the analog. Thereafter the gonadotropin levels fell, but nevertheless remained above those of controls for most of the cycle. Hence, it appears that enhanced follicular growth attributed to the early transient rises in gonadotropins can be coupled to a suppression of endogenous LH surges in leuprolide-treated women. These beneficial effects seem to be more likely to occur if leuprolide is initiated on cycle day 3 rather than day 1.

Cardiac output and blood flow distribution to the genital organs of rabbits was studied using radioactive microspheres for a period of 9 h following HCG administration. Throughout this period the ovarian blood flow was higher (1.2 ± 0.3 mI/mm/organ) than in the estrous animal (0.17 ± 0.02). The maximal ovarian blood flow occurred 4 h after HCG, and was associated with high perfusion rate (12 ± 3 ml/min/g). The cardiac output increased from 276 ± 62 mI/mm in estrus animals to 479 ± 82 following HCG, with a peak value of 780 ± 133 at 4 h. The fractional distribution of the cardiac output to the oviducts (0.18 ± 0.02 percent); uterus (0.40 ± 0.06 percent); brain, (1 percent); and kidney (21 percent) did not change during the preovulatory period. The fraction of cardiac output distributed to the ovaries increased from 0.10 ± 0.3 percent in estrus, to a maximum of 0.43 ± 0.06 percent at 4 h. The differences in the vascular response of the genital organs suggest the existence of local regulatory mechanisms which may play a role in the mechanisms leading to ovulation.

Hormonal profiles were compared in 14 ovulatory women who were treated with two different doses of gonadotropins in successive in vitro fertilization cycles. All patients suffered from mechanical causes of infertility. Serum estradiol (E 2 ), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and progesterone (P) were measured daily during the follicular phase. Women were arbitrarily classified as high responders (E 2 >1000pg/ml on the day of human chorionic gonadotropin administration, n=8) or as low responders (E 2 2 levels during the cycle when they received 3 ampules of human menopausal gonadotropin (hMG). When patients were treated with 3 ampules of hMG, serum FSH, LH, and P concentrations increased significantly during the follicular phase in high responders but remained unchanged in low responders. When these patients were treated with 2 ampules of hMG, the temporal profiles of the hormones were similar, but the magnitude of increases in serum levels of gonadotropins and sex steroids was significantly reduced in high responders. The authors conclude that temporal individuality of endocrine profiles cannot be altered by varying the dose of gonadotropin. Increases in hormone levels accompanying a high response to hMG can, however, be dampened by lowering the dose. In contrast, hormone concentrations are not influenced by changing the dose of hMG in low responders.

The effects of cortisol on steroid secretion by human luteinized granulosa cells was investigated by culturing cells from mature follicles of gonadotropin-treated women. After culturing for 24 hours in medium containing 10% fetal bovine serum, the cells were washed and incubated in a chemically defined medium. Cortisol (10(-5), 10(-6), and 10(-7) M), follicle-stimulating hormone (FSH; 100 ng/ml), FSH plus cortisol (10(-6], or vehicle were added to replicate cultures. After incubating for 6 hours, medium was harvested and assayed for estradiol (E2) and progesterone (P). Cortisol (10(-5), 10(-6) but not 10(-7) M) significantly (P less than 0.05) stimulated E2 secretion when compared to controls. FSH alone did not alter E2 secretion while FSH plus cortisol had a similar effect to cortisol alone. Likewise, P secretion was significantly (P less than 0.05) increased by cortisol when compared with controls and the stimulation was dose dependent. FSH also stimulated P secretion, but the combination of FSH and cortisol did not give an additive or synergistic effect. It appears that cortisol can stimulate the secretion of E2 and P by human granulosa cells without involving FSH receptor interaction. The findings suggest that, in addition to pituitary and ovarian hormones, cortisol may also be directly involved in granulosa cell function.

A radioimmunoassay using an antibody against llα,20α-dihydroxypregn-4-en-3-one-11-succinyl-BSA is described for the measurement of 20α-hydroxypregn-4-en-3-one in human plasma. Sephadex LH-20 column chromatography is used to separate progesterone from 20α-OHP. The sensitivity of the assay is 50 pg. The interassay and intra-assay coefficients of variation were 15% and 10%, respectively. Daily measurement of 20α-OHP throughout the normal menstrual cycle revealed levels of 0.41 ± 0.05 ng/ml throughout the follicular phase, followed by a slow rise after ovulation reaching peak levels of 3.4 ± 0.5 ng/ml in the midluteal phase. A constant ratio of 20α-OHP/P of 0.15 to 0.22 was found throughout the luteal phase. Levels in men were not significantly different from those in the early follicular phase of the normal menstrual cycle.

Collagenase-dispersed cells from superovulated rat ovaries were studied to determine whether LH induces acute changes in ovarian polar lipid metabolism. Exposure of cell suspensions to LH (1 μg/ml) for 2 h caused no change in cell lipid phosphorus content. In vitro incorporation of 32P, [U-14C]glucose and [l-14C]oleate into the phospholipid fractions of cells was not altered by LH, but a small, significant increase in [U-14C]acetate incorporation into total polar lipids was induced by LH. In all studies with labeled compounds, phosphatidylcholine, the predominant phospholipid in superovulated rat ovaries, was most heavily labeled. With 14C-labeled acetate, oleate and glucose, phosphatidylethanolamine, the second most prominent phospholipid, was the second most labeled class, but with 32P, the phosphatidylinositol/phosphatidylserine fraction contained the second greatest radioactivity. LH had no effect upon distribution of 32P, [U-14C]glucose or [l-14C]oleate into various phospholipids. LH produced small i...

Alternate protein sources have been suggested to replace the commonly used cord or patient serum for in vitro fertilization (IVF) procedures. During an 11-month period 127 patients treated for in vitro fertilization had either their serum (N = 71) or bovine serum albumin (BSA; N = 56) used as the protein source in the insemination and growth media. Ham's F-10 + 0.5% BSA was used for sperm swim-up and insemination media and 1% BSA was used for the growth media. Patient's serum was added to Ham's F-10 culture media at concentrations of 7.5 and 15% for insemination and growth, respectively. Embryo transfer was performed with Ham's F-10 containing 90% maternal serum in both groups. Fertilization rate of 259 oocytes inseminated in medium containing patient's serum did not differ when compared with 200 oocytes inseminated in medium containing BSA. Likewise, rates of abnormal fertilization, cleavage, and pregnancy were similar in both groups. In a second experiment, 148 normally fertilized oocytes were transferred after 24 hr in culture to growth media containing two different concentrations of BSA (0.5 or 1%). Cleavage rates for the two groups were similar and the percentage of embryos developed to greater than or equal to 4 cells did not differ significantly. We conclude that a single concentration of BSA can safely be used to supplement culture media in human IVF with several practical and economical benefits.

Sex hormone-binding globulin (SHBG), percent free estradiol (E2), the fraction of E2 bound to SHBG, and total E2 were measured in the serum and follicular fluid of 12 women (25 follicles) who had received gonadotropin stimulation in an in vitro fertilization program. The women were classified as high or low responders based on peak serum E2 levels (high responders: peak E2, greater than 1500 pg/ml; low responders: peak E2, less than 1000 pg/ml). During treatment, serum levels of SHBG increased in high responders from 55 +/- 8.8 (+/- SEM) to 96 +/- 16 nM (P less than 0.01), but did not change in low responders. SHBG was more concentrated in follicular fluids from high responders (142 +/- 12.5 nM) than in those from low responders (44.4 +/- 5.8 nM). A positive correlation was found between serum and follicular fluid levels of SHBG (r = 0.873; P less than 0.01). In follicular fluid, total E2 levels, which varied from 100-2650 ng/ml, correlated (r = 0.790; P less than 0.01) closely with SHBG levels. The percent free E2 averaged 5.9% (range, 4-10.6%) in follicular fluid compared to 1.8% (range, 1.5-2.1%) in serum. An inverse correlation (r = -0.661; P less than 0.01) was found between total E2 concentrations and percent free E2 in follicular fluid. The relationship between serum and follicular fluid levels of SHBG suggests that SHBG in follicles arises from the circulation. Although SHBG is present in follicular fluid in amounts similar to those in serum, the large quantities of E2 in preovulatory follicules exceed the binding capacity for SHBG, and the majority of E2 appears to be bound to albumin. Hence, it seems unlikely that SHBG in follicular fluid regulates estrogen action in ovarian target cells.

Follicular fluid (FF) levels of prolactin (PRL), estradiol (E2), progesterone (P), and androstenedione (delta 4A) were related to diversities in oocyte maturation, fertilization, and cleavage among oocytes obtained for in vitro fertilization. Follicles with mature stage I oocytes contained similar concentrations of PRL and E2, more P, and less delta 4A compared to follicles containing immature oocytes. Follicles with mature stage II oocytes had higher amounts of P and E2 but similar levels of PRL and delta 4A compared to follicles with mature stage I oocytes. Prolactin was lower and E2 higher in FF associated with fertilizable oocytes than in those with unfertilized gametes. Follicles which yielded gametes that ultimately divided to more than four cells had the highest E2 levels, while the lowest E2 concentrations were associated with fertilized, uncleaved eggs. P, delta 4A, and PRL levels in FF were not related to oocyte cleavage. We conclude that human menopausal gonadotropin (hMG) treatment does not necessarily lead to a synchrony or uniformity of preovulatory follicles, maturation of oocytes is associated with rising levels of progesterone and decreasing levels of delta 4A in FF, an increased cleavage rate in vitro is associated with higher FF levels of E2, and high levels of PRL in FF are associated with unfertilized oocytes. These findings emphasize that differences in the hormonal milieu surrounding oocytes may have profound effects on the success of in vitro fertilization.

Iv injections of 75 IU human chorionic gonadotropin were given to New Zealand white rabbits. Ovarian blood flow was measured by using the radiolabeled microspheres technique. Both ovarian veins were cannulated and peripheral blood was obtained simultaneously. Ovarian secretion of hormones was determined at hourly intervals for 9 hours. Ovarian vein plasma concentrations of 17beta-estradiol (E2) and testosterone (T) rose considerably by 2 hours. By 4 hours the secretion rate of E2 reached a 2nd peak. Secretion rates of estrone (E1) androstenedione (A) progesterone (P) and 20alpha-dihydroprogesterone (20alpha-OH-P) showed a peak only at the 4-hour period. Peripheral blood levels of E1 E2 and A were unchanged. A small rise in T between the 2nd-4th hours was noted. An increase in P was sustained from the 1st up to the 8th hour. 20alpha-OH-P was elevated from the 2nd to the 9th hour. Removal rate of HCG was most during the 2nd hour. Ovarian steroidogenesis seemed to be stimulated by HCG. Significant but brief rises in E2 and T production may serve an intraovarian function. Ovarian steroid production observed agrees with that which occurs shortly after mating in rabbits. Cellular binding sites may be involved.

Secretion of progesterone by granulosa cells from preovulatory follicles of mice was determined during 2 weeks of cell culture in the presence of androgens, estrogen and pituitary gonadotropins. Androstenedione (10’ M) and dihydrotestosterone (10W’ M) stimulated ([‘ 0.10) progesterone secretion throughout the culture period. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) stimulated (P

This study compares outcomes of in vitro fertilization (IVF) in two groups of 57 patients when either 2 (group 1) or 3 (group 2) ampules of human menopausal gonadotropin (hMG) were administered daily. Treatment began on day 3 of the cycle and was discontinued when at least 2 follicles attained diameters greater than or equal to 1.5 cm. Human chorionic gonadotropin (hCG) was given either 24 or 48 hours after the last dose of hMG. Although serum estradiol levels were lower in group 1, the average number of oocytes retrieved (3.2 versus 2.9), fertilized (1.9 versus 2.0), and cleaved (1.7 versus 1.8) per completed cycle did not differ between groups 1 and 2. Likewise, the number of oocytes that fertilized abnormally was similar in both groups (0.5 versus 0.3/cycle). However, the number of atretic oocytes (0.03 versus 0.5/cycle) and the percent of oocytes recovered from the cul-de-sac (0 versus 7.2%) were significantly (P less than 0.05) lower in group 1. In group 1, administration of hCG 48 hours after the last dose of hMG was associated with a higher number of cleaving embryos (2.1 versus 1.5/cycle) and a higher pregnancy rate (34.8 versus 14.7%; P less than 0.05) when compared with injection at 24 hours. In group 2, the interval between hMG and hCG did not influence these results. Together, the associations between fewer oocytes that were atretic or recovered from the cul-de-sac, and a trend toward a higher pregnancy rate, suggest that follicular recruitment with 2 ampules of hMG is more appropriate than 3 ampules in an IVF program.

Progesterone binding of high affinity with a dissociation constant of 10(-9) M was identified in cytosol of rabbit oviduct and uterus. Macromolecules with sedimentation coefficients of 7-8 S and 4-5 S were present. Progesterone receptor concentration was two to fivefold lower in the oviduct when compared with the uterus. The receptor concentration declined steadily from 3 hr until 144 hr after mating in the uterus; however, the decline in oviductal receptor was not significant until the sixth day of pregnancy. Progesterone receptor concentration in rabbit oviduct and uterus in estrus and early pregnancy was greater than estradiol receptor levels.

A progesterone binding protein was characterized in the cytosol of human myometrium. The receptor sedimented in the 4S region on sucrose gradients and had an equilibrium dissociation constant of 3.7 times 10-9M. Synthetic progestagens had an affinity equivalent to that of progesterone while cortisol had low affinity for the binding protein. These same progestagens showed very low binding to human CBG. In normally cycling women, the total levels of cytosol receptor were higher during the follicular phase than during the luteal phase. Subjects taking oral contraceptives and those who were pregnant had the lowest concentration of myometrial progesterone receptor. Postmenopausal women had levels of myometrial receptor lower than those of the secretory phase of the cycle. Estrogen therapy increased the receptor concentrations of the menopausal myometrium to those of the proliferative phase. The estradiol receptor levels of myometrial cytosol revealed similar changes in cycling women. When both binding proteins were measured concurrently in the same specimen, there was a significant positive correlation.

We measured the mitotic activity of granulosa cells, sex steroid concentrations in follicular fluids, and the maturity and fertilizability of oocytes from 49 follicles. Flow cytometric measurements of DNA were used to determine the percentage of cells in G0/G1, S, and G2/M phases of the cell cycle. Mitotic index was designated as the percentage of granulosa cells in S + G2/M. The progesterone concentration and the progesterone to estradiol ratio in follicular fluids were inversely correlated to mitotic index (r = -0.506; P less than 0.001, and r = -0.320; P less than 0.02, respectively). Estradiol and androstenedione levels did not correlate with the mitotic index. The mitotic index was higher in follicles with immature oocytes [25.6 +/- 2.0% (+/- SE); n = 7] than in follicles with mature oocytes (15.6 +/- 1.2%; n = 41; P less than 0.001). The mitotic index of granulosa cells was lowest in follicles with oocytes that fertilized (15.5 +/- 1.8%), higher in follicles with oocytes that remained unfertilized (18.5 +/- 1.3%; P less than 0.03), and highest in follicles with oocytes that fertilized abnormally (24.0 +/- 2.1%; P less than 0.02). Differences in maturity or fertilizability of oocytes were not associated with variations in follicular fluid progesterone concentrations. The study supports the concept that mitotic activity is decreased when granulosa cells become luteinized. During early follicular growth it is assumed that estradiol and perhaps androstenedione may be important regulators of cell division. Our findings suggest that progesterone, perhaps acting as an antiestradiol, is more important in controlling granulosa cell division of preovulatory follicles during the late follicular phase.

Sex hormone-binding globulin (SHBG), estradiol (E2), percent free E2, percent of E2 bound to SHBG, and testosterone (T) were evaluated in 28 ovulatory women during human menopausal gonadotropin-stimulated cycles for in vitro fertilization. Patients were divided into two categories: low responders, in whom serum E2 concentration reached levels less than 1000 pg/ml (mean, 638 +/- 93), and high responders, with serum E2 levels greater than 1000 pg/ml (mean, 2219 +/- 330). A significant increase in SHBG can occur within a short time in high responders (from 62.8 to 103.9 nmol/l) but not in low responders. This increase is accompanied by a significant decrease in the percent free (bioavailable) E2, but the distribution of E2 between the fraction bound to SHBG or albumin did not vary. Despite the increase in the levels of SHBG, the concentration of bioavailable (free) E2 in hyperstimulated women is higher than in normal cycles. The significant increase in T in high responders, by virtue of its higher affinity for SHBG, probably contributes to the increased levels of bioavailable E2.

Estrogen receptor levels of cytosol and nuclei were measured in the cervix, vagina, myometrium, and endometrium of the rhesus monkey after the following experimental treatments: (1) castration; (2) castration with estradiol treatment; and (3) castration with estradiol plus progesterone treatment. Plasma hormone levels similar to those of normal menstrual cycles were achieved by implantation of silastic capsules containing estradiol or progesterone. The K D , 4 × 10 −9 M, for the cytosol receptor was similar in all tissues. Competition studies showed cytosol receptor affinities to be E 2 > e 1 > E 3 > DES. Cytosol receptor levels in estrogenized animals were lowered by progesterone treatment. Progesterone treatment did not alter the levels of nuclear receptor indicating that the lower levels of cytosol receptor obtained with progesterone were not due to nuclear translocation of cytosol receptor. Estrogen treatment changed the sedimentation coefficient of the endomentrial and myometrial cytosol receptor from 4 s to 8 s when compared with the castrate. This alteration in sedimentation coefficient was blocked by progesterone treatment. Changes in sedimentation coefficient were not observed in the cervix or vagina. Nuclear receptor in estrogen and estrogen plus progesterone treated animals sedimented as a 5 s protein. The data suggest that both qualitative and quantitative changes in the estradiol cytosol receptor of the genital tract are induced by progesterone and that these alterations may be related to the hormone interactions in target organs.

The levels of estrogen receptor in the cytosol of different segments of the human fallopian tube obtained at different times in the reproductive cycle were measured in 38 20-48 year old women undergoing surgery for pelvic disease. Estradiol binding was in the 4S and 8S regions. Excess unlabeled estradiol displaced most radioactivity from the 8S area with little effect on the 4S area. The dissociation constant of the receptor was 1.1 times 10 less than minus 9 greater than. The greatest amount of binding was seen at the ampulla with the lowest in the infundibulum. Adjacent to the corpus luteum there was an even lower level of estradiol receptor. These differences were not apparent in women taking oral contraceptives.

The changes in estradiol binding in the cytosol of tubal and uterine tissues of rabbits at different times after mating are described. The results are compared with receptor activity in tissues from castrated and estrous animals. Estradiol binding time is affected by incubation temperature. Estradiol binding was found in the 4S and 8S regions by analysis of sucrose gradients of uterine and tubal cytosols. 3 hours after coitus the level of receptor was not different from the estrous state. However 12 hours after coitus a significant decline in concentration had occurred in both tissues. 3 days after mating the levels increased again then decreased to the lowest levels at 6 days postcoitus. The changes in estradiol receptor concentrations seem to be related to variations in ovarian hormone production and tubal contractility from the time of mating to implantation and may thus be important in regulating tubal and uterine function.(AUTHORS MODIFIED)

The role of blood cholesterol in ovarian function was studied by treating superovulated rats with 4-aminopyrazolo [3,4,-cd]pyrimidine (APP) a drug which inhibits hepatic secretion of lipoproteins. Reduction of plasma cholesterol and progesterone concentrations and ovarian content of cholesteryl esters were related to dose and duration of APP treatment; ovarian concentrations of free cholesterol, phospholipids, and triglycerides and ovarian weight were not altered. Progestin content of luteal tissue was also reduced by APP treatment. These changes were reversible, since concentrations of plasma progesterone and cholesterol and ovarian concentrations of cholesteryl esters returned to control values 5 days after stopping treatment. Dispersed ovarian cells from APP-treated rats incorporated [14C]- acetate into free cholesterol at a 10-fold greater rate than control cells. Luteal cells from both groups incorporated [14C]acetate into cholesteryl esters, phospholipids, triglycerides, and 14CO2 at similar rates. ...

Methods are described for the measurement of the estradiol-binding capacity of TeBG and of the free, TeBG-bound, and non-specifically protein-bound fractions of plasma estradiol. Each determination used undiluted plasma at 37 C, and a total volume of less than 2.0 ml of plasma was required to complete all the assays. The measurement of the per cent of free estradiol was affected by changes in plasma dilution. The measurement of the other fractions of estradiol was not influenced by changes in either the dilution or the volume of plasma. The distribution of plasma estradiol was determined daily throughout 5 individual menstrual cycles. The per cent of free, the per cent of TeBG-bound, and the TeBG binding capacity of estradiol remained constant throughout the cycle with mean values of 2.21 +/- 0.04% (SE), 38.4 +/- 0.7%, and 16.6 +/- 0.43 ng/ml, respectively. The mean association constant of TeBG for estradiol was 6.58 +/- 0.25 x 10(7)M-1. The concentration of the free and non-specifically protein-bound fractions of estradiol paralleled the total plasma concentration of estradiol. The results show that biologic events related to normal cyclic changes of plasma estradiol may be attributed to fluctuations in the free estradiol and to estradiol which is bound with low affinity to non-specific plasma proteins.

The effects of social interaction on the adaptive response of the testes were studied by maintaining chickens in experimental heterosexual groups for periods of 12 to 30 weeks. Increases in group size resulted in an increase in social conflict within groups. This interaction was reflected by smaller testes among the grouped males when compared with isolated and heterosexually paired cocks. The number of birds within a group rather than density seemed to be the important factor in determining the intensity of social interactions. Thus, in groups where sufficient space was allotted for freedom of movement, the frequency of interactions and decrease in testicular weight was as great as among groups of the same size where density made social contacts unavoidable. Within the groups testes weights were correlated with social rank. The most dominant males were found to have the heaviest testes while the subordinate cocks had the lightest and, occasionally, atrophic testes. The smaller testes associated with increased group size and subordinate social rank were attributed to degenerative changes in the germinal epithelium of grouped subordinate cocks. The most frequently encountered testicular changes were the formation of polynuclear giant cells and associated degenerative lesions within the germinal epithelium. In a few subordinate males complete regression was observed in the germinal layer of the testes. These observations support the hypotheses that social conflict may be an important factor in determining the adaptive response of the testes of chickens. Certain features of this response suggest that domestic fowl provide a useful species for further studies of reproductive function as related to their characteristic social interaction.

A procedure for the direct transfer of steroids from one TLC to another is described. This method eliminates solvent elution and evaporation between successive chromatographies. As a result, the recovery of certain steroids is improved and less time is required for repeated TLC of a large number of samples.

A corpus luteum, placenta, and placenta plus foetal viscera of a 13-week old human pregnancy were incubated with dehydroepiandrosterone-7α-3H in vitro. Conversion to oestrone and 17β-oestradiol was found in all incubates. Oestriol was formed from dehydroepiandrosterone only in the incubate of placenta plus foetal viscera. Thus, in early pregnancy dehydroepiandrosterone may serve as a precursor for oestrogen biosynthesis not only in the foeto-placental compartment, but also in the corpus luteum.

SummaryThe incorporation and fate of estradiol-17β-6,7-H3 was studied in the female rabbit. The Fallopian tube of the rabbit showed an affinity for estradiol-17β at the same level as uterine tissue. The major portion of radioactive steroids present in the Fallopian tube, uterus and skeletal muscle was in the ether-soluble fraction, whereas in plasma significant amounts of activity were present in the aqueous layer. Estradiol-17β was the only radioactive estrogen found in the tubal and uterine tissues. Radioactivity was identified as estrone and estradiol-17β in the skeletal muscle, and as estrone, estradiol-17β and estriol in the plasma.

Incubation of two corpora lutea of early human pregnancy in the presence of dehydroepiandrosterone-7α-3H resulted in a 33—44% yield of oestrone plus 17β-oestradiol. The major oestrogen formed in homogenate preparations was oestrone while in a slice preparation it was 17β-oestradiol. Oestrone and 17β-oestradiol were characterized by solvent partition, paper chromatography and crystallization to constant specific activity after addition of carrier. Acetates of the crystallized oestrogens had the same specific activities as the parent steroids and these derivatives were further identified by thin layer chromatography. These results suggest that dehydroepiandrosterone may serve as a major precursor for oestrogen biosynthesis by human corpus luteum of early pregnancy.

The average blood flow to the ovaries, fallopian tubes, uterine horns and vagina of estrus rabbits was 1.40 ± 0.14, 1.44 ± 0.13, 0.75 ± 0.06, 0.30 ± 0.04 ml/min/g of tissue, respectively. There was no differences between the right and left organs. Ligation of the ovarian artery resulted in 80–90% decrease in blood flow to the ovary and oviduct, but recovery occurred within 1 and 6 days for the ovary and oviduct, respectively. There was no effect on the uterus and vagina. Ovulatory response to HCG occurred within 1 day and pregnancy after 6 days following ligation.

At the University of Pennsylvania School of Medicine in Philadelphia the electrophoretic patterns and protein binding of steroids in rabbit uterine and oviductal fluids were investigated. Use was made of polyacrylamide gel electrophoresis. A postalbumin (migrating behind albumin) protein appeared in the uterine fluid after ovulation. This proteins concentration increased during early pregnancy or pseudopregnancy reached a maximum on the fifth day postovulation and decreased to nearly 0 by the ninth day postovulation. The proten was absent in serum and in oviductal fluid of pregnant and pseudopregnant rabbits. Progesterone or progesterone and estradiol administration resulted in the appearance of this protein in the uterine fluid of oophorectomized females but failed to induce its appearance in the oviductal fluid of these animals. In oophorectomized females receiving only estradiol the uterine or oviductal patterns were unaffected. The postalbumin protein which migrated in front of transferrin and whose appearance was obviously stimulated by progesterone has a significance yet unclear.

Radiolabeled estrogens were isolated from the urine of two rhesus monkeys after iv injections of estrone–6,7–3H and estradiol–17β–6, 7–3H. The injection was given on day 12 of the individual menstrual cycle. The following estrogen metabolites were isolated from the urine: estrone (E1) estradiol–17β (E2), estriol (E3) and 16–epiestriol (16–epi E3). In both animals, estrone was found to be the major urinary metabolite during the first 24–hr collection period. On days 2, 3, 4, and 5 after the injection, the relative amount of E1 decreased while the 16–epi E3 fraction was increasing to levels of 33–52% of the identified estrogens. The 16–epi E3 was identified by four thin—layer chromatographies, one paper chromatography, two derivative formations and recrystalization to constant specific activity. It is concluded that the metabolism of E1 and E2 to 16–epi E3 is a major pathway in the rhesus monkey. This conclusion is supported by the determination of endogenous levels of this urinary estrogen fraction in a go...

A macrophage homogenate contained substances which stimulated primary cultures of mouse granulosa cells to secrete more progesterone. The response to the luteotropic substances was similar to that observed when intact macrophages were co-cultured with granulosa cells. The bioactive polypeptides present in cytosolic and particulate fractions of cell homogenates were non-dialyzable, heat labile and trypsin sensitive. When the surface of intact macrophages was treated with trypsin there was a loss of activity from the particulate fraction suggesting that some luteotropic proteins reside on the plasma membrane of mononuclear phagocytes. Treatment of macrophages with Con A but not the succinyl derivative of the lectin caused a release of luteotropic proteins with apparent molecular weights of 26,000 and 41,000. These findings in conjunction with our prior observation that macrophages must contact granulosa cells to stimulate progesterone secretion suggest that aggregation of mononuclear cell surface proteins may occur when the two cells interact thus resulting in the expression of luteotropic activity. Hence, it appears that macrophages which are found within corpus luteum may be a source of ovarian cybernins. This is the first description that a cell of the immune system can communicate at the molecular level with a steroid secreting cell of the ovary.

Increasing levels of E2 in gonadotropin-treated women stimulated hepatic synthesis of SHBG and TBG, and consequently increases in T4 concentration. Nevertheless, unchanged FTI and TSH suggested that a euthyroid state was maintained. The temporal patterns for the rise in serum concentrations of TBG and SHBG during gonadotropin therapy suggest that the synthesis of these proteins by the liver has different sensitivities to E2.

A profound suppression of the pituitary and the ovary is connected to an inadequate response to gonadotropin therapy. The therapy used to produce multiple follicular development for in vitro fertilization (IVF) can cause a variety of responses including: premature luteinization inadequate amount of preovulatory follicles and an asynchrony of follicular maturation. The aforementioned responses are related to lowered IVF success. IVF patients who received oral contraceptives as part of treatment had undesirable responses to human menopausal gonadotropin when the contraceptives were administered for only a short period. It is suggested that a different regimen be pursued to achieve adequate responses. A pretreatment determination of E and gonadotropins is a viable method of predicting ovarian response among women who have suppressed pituitary-ovarian function. It is further concluded that a favorable response to hMG was not found in IVF patients when treatments of oral contraceptives are administered for a short period.

Several solvent systems are described for the separation of neutral and phenolic steroids. Benzene and methanol in different proportions, with and without methylene chloride or chloroform, were found to be the most suitable systems for the separation of the 3 classic estrogens. Heptane, isooctane, hexane and cyclohexane were the basic solvents used for the chromatography of various neutral steroids. These systems required the addition of small amounts of either benzene, ethyl acetate or chloroform together with methanol. Sephadex LH-20 column chromatography offers several features which make it particularly useful in steroid assay procedures such as competitive protein binding and radioimmunoassay.

A specific progesterone antibody was obtained by immunizing a ewe with 11α-hydroxy-progesterone-hemisuccinate conjugated to bovine serum albumin. This antibody was used for the assay of progesterone in crude plasma extracts. The sensitivity of the assay was 0.05 ng. The accuracy was confirmed by the recovery of known amounts of progesterone added to plasma. The inter- and intra-assay coefficient of variation was 8%. Specificity was confirmed by a) checking cross reactivity of various steroids; b) failure of other steroids added to plasma to influence the accurate measurement of progesterone, and c) finding comparable progesterone levels in plasma extracts with and without chromatography. Mean values of progesterone (ng/ml) were 0.26 ± 0.08 (sd) in males and postmenopausal females; 0.50 ± 0.44 in preovulatory females; and 12.1 ± 4.81 in postovulatory females.

To study the role of lysosomes in ovarian function, we examined effects of lysosomotropic drugs on progesterone secretion by suspensions of cells prepared from superovulated immature rat ovaries. 50 μM chloroquine (CQ) produced a 45% inhibition of progestin secretion by LH-treated cell suspensions. The effect of CQ was dose-dependent with 30% and 60% inhibition of the secretory response to LH being caused by CQ concentrations of 25 μM and 200 μM, respectively. CQ (50 μM) also partially blocked the steroidogenic response of ovarian cells to dibutyryl cAMP. Incubation of dispersed ovarian cells in the presence of neutral red (0.345 mM), NH 4 Cl (10 mM) or Triton WR-1339 (0.75%) also resulted in partial inhibition of progesterone secretion in response to LH. CQ (50 μM) did not alter incorporation of [4,5- 3 H]-leucine into protein, [U- 14 C]-glucose oxidation, or lactate production by the ovarian cell suspensions. Furthermore, CQ did not inhibit formation of [ 14 C]-pregnenolone and [ 14 C]-progesterone by ovarian mitochondria incubated in the presence of 5 mM succinate and [4- 14 C]-cholesterol. Many of the cells exposed to CQ developed large vacuoles during a 1 h incubation. These membrane-enclosed vacuoles contained amorphous material and membrane whorls. Although one must be cautious in assigning specific loci of action to inhibitors, our observations on the effects of several lysosomotropic drugs on progesterone secretion by LH-stimulated ovarian cells suggest that lysosomes are involved in the process of steroidogenesis.

A specific progesterone receptor of myometrial cytosol from the rhesus monky is described. Characterization of the receptor by sucrose density gradient centrifugation revealed 2 peaks at 4s and 7.5s. The 4s peak seen in all groups (castrate; castrate plus estrogen treated; castrate plus estrogen and progesterone treated) contained little specific progesterone binding but the 7.5s peak, seen only in the estrogen-treated animal, was specific for progesterone. Competition studies revealed the reeceptor affinities to be: progesterone 100, 5alpha dehydroprogesterone 81.9 melengestrol acetate 72.5, norgestrel 53, desoxycorticosterone 25.9, 5beta-dihydroprogesterone 1.2, and 17 hydroxyprogesterone less than 1. Receptor levels measured from Scatchard plot analysis of of equilibrium data were 7 fM/mg cytosol protein (castrate), 45.2 fM/mg (p less than .01, estrogen treated), and 10.5 (estrogen plus progesterone treated). The association constant (approximately 5 x 10(-9)M) was similar in all 3 groups.

To further investigate prolactin (PRL) secretion in polycystic ovary syndrome (PCO), the authors evaluated immunoreactive (immuno) and bioactive (bio) PRL levels in the basal state and in response to provocative testing with intravenous dopamine (DA), metoclopramide (MCP), and gonadotropin-releasing hormone (GnRH), before and after disulfiram. Basal measurements of immuno-PRL, bio-PRL, and the ratio of bio/immuno-PRL were similar in PCO and controls. The immuno-PRL decrement after DA was greater than that of bio-PRL in both groups (P less than 0.05). After MCP, immuno-PRL increased more than bio-PRL in PCO (P less than 0.01), and this immuno-PRL increment was greater than that of controls (P less than 0.05). Bio-PRL and immuno-PRL increased after GnRH in PCO, but not controls, and these responses were inhibited by disulfiram. These data confirm PRL hypersecretion in some women with PCO, which is better expressed by immunoreactivity than bioactivity. Given the assay systems and patients studied, bioactivity

The temporal changes of estradiol levels in 19 women undergoing ovulation induction for in vitro fertilization (IVF) and embryo replacement were described by a mathematical model. The model was analyzed for differences between treatment cycles of an individual woman and between cycles of different women. This model was also used to evaluate the results of IVF treatment. The variation between cycles within individuals was found to be less than that between different women. The parameters that describe this model were found to correlate with follicular growth but not with number of oocytes retrieved, fertilized or cleaved.

Cells with the ultrastructural features of macrophages were seen within corpora lutes of mice shortly after induction of ovulation with gonadotropins. Upon isolation, mononuclear phagocytes from luteal tissue had histochemical and functional properties characteristic of macrophages from other body sites. These attributes included intense staining for nonspecific esterase activity, phagocytosis of lgG-coated red blood cells via an Fc receptor, ingestion of latex beads, and trypsinresistant adherence to the surface of culture dishes. Furthermore, the ovarian macrophages specifically bound to their surface a monoclonal antibody against a plasma membrane protein of mononuclear phagocytes. When mouse granulosa cells were co-cultured with peritoneal macrophages, progesterone secretion was increased, and the magnitude of the increased steroidogenesis was directly related to the number of mononuclear phagocytes added to the cultures. Likewise, cultured luteal cells from superovulated mice secreted more progestins in the presence of peritoneal macrophages. Luteal cells also produced more progesterone when they were recombined in culture with autologous macrophages from corpora lutea. The mononuclear phagocytes from ovarian tissue appeared to have a greater ability to stimulate luteal cells than did peritoneal macrophages. Conditioned media from cultured macrophages failed to stimulate steroid secretion by granulosa cells, suggesting that close proximity of the two cells is required for the effect on luteal cell function. Thus, it appears that macrophages within corpora lutea may play a role in maintaining progesterone secretion by luteal cells.

Estrogen binding in endometrial tissue of various hormonal conditions was compared by a quantitative dextran-coated charcoal assay. The amount of [ 3 H]E 2 bound to cytosol in vitro was expressed as fmole/ μ g DNA and fmole/mg protein. The highest values were obtained for proliferative endometrium (1.95 ± 0.45; 244.06 ± 38.99). Endometrium from patients on oral contraceptives had low levels of estrogen receptor, which was slightly higher than that of atrophic endometrium. Various endometrial lesions were examined. The most consistent results were found for areas of hyperplastic endometrium (1.51 ± 0.41; 138.88 ± 3.43). Seventeen cases of endometrial adenocarcinoma plus one case of endocervical adenosquamous carcinoma can be divided into three groups. One-third showed no detectable estrogen binding, one group of seven patients showed low values, and a third group of five patients had high concentrations of estrogen receptors.

1. 1. Lysosome-enriched fractions were prepared by differential centrifugation of homogenates of luteinized rat ovaries. Acid phospholipase A activities were characterized with [U- 14 C]diacyl- sn -glycero-3-phosphocholine and 1-palmitoyl-2-[9, 10- 3 H]- or [1- 14 C]oleoyl- sn -glycero-3-phosphocholine as substrates. Acid phospholipase A 1 activity had properties similar to other hydrolases of lysosomal origin; subcellular distribution, latency and acidic pH optimum. Acid phospholipase A 2 activity with similar characteristics was also tentatively identified. We were unable to exclude the possibility that the combined action of phospholipase A 1 and lysophospholipase contributed to the release of acyl moieties from the 2-position of the synthetic substrates. 2. 2. Lysophospholipase activity was present in the lysosome-enriched fractions. This activity had an alkaline pH optimum. 3. 3. Phospholipase a 1 and A 2 activities solubilized from lysosome fractions by freeze-thawing were inhibited by Ca 2+ and slightly activated by EDTA. A Ca 2+ -stimulated phospholipase A 2 activity, with an alkaline pH optimum, remained in the particulate residue of freeze-thawed lysosome preparations. This activity is believed to represent mitochondrial contamination. 4. 4. Activities of acid phospholipase A, as well as other acid hydrolases, increased approx. 1.5-fold between 1 and 4 days following induction of luteinization, suggesting a hormonal influence on lysosomal enzyme activities.

The role of prolactin (PRL) in the regulation of human ovarian function is poorly understood, and reported findings are often contradictory. We have evaluated the effects of several doses of PRL on the secretion of progesterone (P) and estradiol (E 2 ) by human granulosa cells from preovulatory follicles of gonadotropin-stimulated women. The effects of PRL were also evaluated when androstenedione ( Δ 4 A) was added to the cultured cells. Our findings show that the human granulosa lutein cells retain their ability to secrete large amounts of P and lesser quantitities of E 2 for a prolonged period in culture. E 2 , but not P, secretion was stimulated by the presence of Δ 4 A. PRL in low doses (25 and 50 ng/ml) did not alter the secretion of either E 2 or P. In contrast, high doses of PRL (100 and 1000 ng/ml) resulted in a significant suppression of E 2 secretion. P secretion was inhibited only by the highest dose (1000 ng/ml) of PRL. We conclude that steroidogenesis by the granulosa lutein cells of preovulatory human follicles is probably not influenced by the quantitities of PRL that are normally present in the blood or follicular fluid. Higher doses, however, may suppress production of E 2 and P.

Steroid secretion and structure of granulosa cells on floating collagen gels were compared with those of cells grown on plastic. Granulosa cells from follicles of gonadotropin-treated women were plated either onto dishes coated with type I collagen or onto plastic dishes. Medium containing serum was removed after 24 hours, defined medium was added, and the gel was floated. Medium was changed daily for 3 days, after which the granulosa cells were prepared for light and electron microscopy. Cells grown on collagen secreted significantly more estradiol and progesterone than those grown on plastic during the 3 days of culture. The round multilayered granulosa cells on collagen had abundant mitochondria and lipid droplets and they formed numerous intercellular junctions. On plastic surfaces, flat granulosa cells grew as a monolayer with few junctions and less abundant mitochondria or lipid droplets. We conclude that growth on floating collagen promotes structural changes of human granulosa cells that enhances cell interaction and secretion of steroid hormones.

Similar incidences of congenital abnormalities in in vitro fertilization and embryo transfer (IVF-ET) pregnancies and births in the general population have not supported earlier concerns that IVF may increase chromosomal aberrations. Nevertheless, polypronuclear fertilization is a common, undesirable, and poorly understood outcome of IVF. We evaluated hormone levels in 20 follicular fluids that were associated with mature oocytes that have fertilized abnormally (greater than or equal to pronuclei) and compared them to follicles with mature oocytes that cleaved normally, fertilized but failed to cleave, or did not fertilize. Progesterone (P), androstenedione, estradiol, percent free estradiol, sex hormone-binding globulin, insulin, and prolactin were measured. P levels were significantly higher in follicular fluids associated with oocytes that fertilized abnormally than in the other groups. Levels of the other hormones were similar in all the study groups. We conclude that IVF of oocytes from highly luteinized follicles as judged by P levels may result in polypronuclear fertilization.

Follicular maturation parameters predictive of decreased success in human in vitro fertilization were evaluated in 104 gonadotropin-stimulated cycles. Falling estradiol (E 2 ) levels correlated with decreased fertilization (1.9 oocyte/cycle) and cleavage (1.4 oocyte/cycle), when compared with cycles with increasing E 2 (2.4 and 2.1 oocyte/cycle, respectively). Likewise, polyspermic fertilization was higher in the former group. Falling E 2 levels after human chorionic gonadotropin (hCG) administration correlated with similar trends. Delaying hCG (24 to 96 hours) relative to the last dose of gonadotropin did not affect the total number of oocytes fertilized per cycle. However, polyspermic fertilization and cancellation rates were higher. Leading follicles growing beyond 2.3cm (determined by ultrasound examination) were associated with decreased recovery, fertilization, and cleavage, and increased polyspermic fertilization. We conclude that ultrasonography and E 2 monitoring can predict decreased fertilization and cleavage and should also be monitored after hCG administration.

Two groups of normal ovulatory women who displayed either a marked (high responders; HR) or a more subtle (low responders; LR) ovarian response to a fixed dose of human menopausal gonadotropins (hMG) were evaluated for differences in blood levels of hormones. Serum follicle-stimulating hormone (FSH) levels doubled during the first 3 days of treatment (to approximately 20 mIU/ml) in all patients; thereafter, the levels plateaued in LR but continued to rise steadily (to 35 mIU/ml) in HR. In the latter group, rise in estradiol (E2) and FSH was accompanied by an increase of luteinizing hormone (LH; two to five times) progesterone (P; four to eight times) testosterone (T; three to four times) and prolactin (PRL; 2 times) toward the end of the follicular phase. Positive correlation was found between FSH and E2 in HR and LR. Positive correlation was found, however, between LH, T, and P and between E2, P, and PRL only in HR. The extent of FSH accumulation in the circulation may be a principal factor in determining an individual's response to hMG therapy. Temporal changes of blood hormones indicated that the continuous rise in FSH levels in HR was associated with early luteinization of the follicles. Increased secretion of P in the follicular phase of these women (HR) probably synergized with the elevated E2 levels to elicit LH release. Similar changes in blood hormones were not found in LR.