The effect of dietary protein on reproduction in the mare. V. Endocrine changes and conception during the early post partum period

Pregnant Anglo-Arab and Thoroughbred mares (n = 24) were divided randomly according to age and breed into 4 groups of 6 mares each from approximately 6 weeks before their expected foaling date. Diets received by the 4 groups varied in essential amino-acid and total protein contents. Serum progestagen, FSH and LH concentrations were determined from the day of parturition until foal heat and during the 1st oestrous cycle following foal heat. Serum progestagen, FSH and LH concentrations did not differ between the treatment groups. Progestagen concentrations were high (mean = 7.0: 5.2-16.4 ng/ml) at parturition but decreased rapidly within 48 h. As progestagen concentrations decreased LH concentrations increased from Days 3-6 post partum to reach maximum values at, or the day after ovulation. FSH concentrations declined 3-4 d after parturition and increased 2-3 d before ovulation at foal heat. The duration of elevated progestagen concentrations during the luteal phase of the subsequent oestrous cycle affected the interovulatory period. A 12-14 d FSH cyclical releasing pattern occurred. Season/photoperiod affected the resumption of normal oestrous cyclicity during the post partum period. The duration of the 1st oestrous cycle after foal heat in mares fed a low-quality protein diet showed a greater range (13-30 d) compared to mares fed a high-quality protein diet (18-26 d).     

Metabolism and excretion of oestradiol-17beta and progesterone in the Sumatran rhinoceros (Dicerorhinus sumatrensis)

3H-labelled oestradiol-17beta and 14C-progesterone were injected i.v. into an adult female Sumatran rhinoceros (Dicerorhinus sumatrensis) and all urine and faeces collected over 4 days. Of the injected steroid, 68% of 3H-oestradiol and 89% of 14C-progesterone were recovered. Peak excretion in urine occurred on day 1 for both steroids, and for faeces on day 2 for 14C-progesterone, and between days 2 and 3 for 3H-oestradiol. Oestradiol metabolites were predominantly (nearly 70%) excreted into the urine, while progesterone metabolites were almost exclusively (> 99%) excreted into the faeces. The majority (> 70%) of urinary excreted oestrogens consisted of water-soluble (i.e., conjugated) forms, with > 90% of these being glucuronides. In contrast, > 75% of faecal oestrogen and progesterone metabolites were excreted as ether-soluble (i.e., unconjugated) forms. HPLC co-chromatography of oestrogens in hydrolysed urine indicated only one peak of radioactivity, co-eluting with authentic oestradiol-17beta, whereas two peaks of radioactivity were found after HPLC of faecal oestrogens, the major one co-eluting with oestrone and the less prominent one with oestradiol-17beta. Progesterone was excreted as numerous metabolites into the faeces. The three most abundant of these were identified using HPLC and gas chromatography mass spectrometry (GCMS) as 5beta-pregnane-3alpha,20alpha-diol, 5beta-pregnane-3alpha-ol-20-one, and a second pregnanediol, the exact structure of which could not be deduced. Measurement of urinary oestradiol-17beta and faecal immunoreactive pregnanediol and 5alpha-pregnane-3alpha-ol-20-one in daily samples enabled the first endocrine characterization of the ovarian cycle and indicated a cycle length of approximately 25 days.     

Comparison of different progestagen assays for measuring progesterone metabolites in faeces of the bitch

From 12 bitches of various breeds with fertile oestrus cycles faecal samples were collected daily from the onset of pro-oestrus till 20 days after the start of vulval bleeding, then once per week till about 1 week before term. Immunoreactive progesterone metabolites were extracted from the samples using methanol and measured using immunoassays. In a first experiment four different assays were compared in regard to the amounts of immunoreactive substances measured: the enzyme immunoassay against 20-oxo-3-hydroxypregnanes showed twice to four times higher values of immunoreactive material than another using an antibody against 6 beta-hydroxyprogesterone. An enzyme immunoassay for pregnanediol measured only low concentrations of immunoreactive material. Also a radio immunoassay using an antibody against 11 beta-hydroxyprogesterone detected only small amounts of reacting material. High performance liquid chromatography showed that in faeces of bitches the immunoreactive progesterone metabolites were present in unconjugated form, mainly as 3 alpha/beta hydroxylated progestagens with a 20-oxo group. In the second experiment the samples were measured with the assay system using the 20-oxo-3-hydroxypregnane antibody. A few days before mating the concentration of progesterone metabolites increased, reaching 5.77 mumol/kg faeces (median) at the day of mating. High levels (10.45 mumol/kg faeces) were measured till the end of the first month after mating. Thereafter, the concentrations decreased, reaching 2.68 mumol/kg (median) at the end of the second month.     

Changes in gonadotrophin response to gonadotrophin releasing hormone in normal women following bilateral ovariectomy

OBJECTIVE: Pituitary responsiveness to GnRH varies throughout the normal menstrual cycle. We have investigated whether there are differences in the ovarian mechanisms which regulate gonadotrophin secretion between the follicular and the luteal phase of the cycle. DESIGN: Normally ovulating women were studied during the first week following hysterectomy plus bilateral ovariectomy performed either in the mid- to late follicular phase (follicle size 16 mm) or in the early to midluteal phase (5 days post LH peak). The response of LH to a single dose of 10 micrograms GnRH was investigated 2 hours before the operation and every 12 hours after the operation until postoperative day 4 and every 24 hours until day 8. PATIENTS: Fourteen normally cycling premenopausal women with normal FSH (< 10 IU/l). Seven women were ovariectomized in the follicular and 7 in the luteal phase. MEASUREMENTS: Pituitary response to GnRH was calculated as the net increase in FSH (delta FSH) and LH (delta LH) at 30 minutes above the basal value. RESULTS: Basal levels of FSH and LH before the operation were significantly lower in the luteal than the follicular phase (P < 0.05), while those of oestradiol (E2) were similar. Also, similar were delta LH and delta FSH values. Serum progesterone and immunoreactive inhibin (Ir-inhibin) concentrations before the operation were higher in the luteal than the follicular phase (P < 0.05). Following the operation, serum E2, progesterone and Ir-inhibin values declined dramatically, while basal FSH and LH as well as delta FSH values showed a gradual and significant increase. The percentage increase in FSH and LH values (mean +/- SEM) on day 8 after the operation was similar in the follicular (453 +/- 99% and 118 +/- 35% respectively) and the luteal phase (480 +/- 71% and 192 +/- 45% respectively). In contrast to delta FSH, delta LH values after a temporal increase 12 hours from the operation, remained stable in the follicular phase and declined significantly in the luteal phase up to day 4. CONCLUSIONS: Basal gonadotrophin secretion during the normal menstrual cycle is predominantly under a negative ovarian effect. It is suggested that in contrast to FSH, the secretion of LH in response to GnRH is controlled by different ovarian mechanisms during the two phases of the menstrual cycle.     

The onset of the ovarian cycle following calving

The onset of ovarian activity post partum was investigated by measuring progesterone concentrations in milk samples, in two dairy herds consisting of 118 cows with an average milk yield of 8340 kg FCM. Samples were taken three times a week till 50 days post partum. In 17 cows (14.4%) anoestrus occurred. The daily milk yield in this group was 2.65 kg FCM higher than the average yield in the group returning to oestrus before day 50 post partum. In cows returning to oestrus within 50 days post partum the first rise in progesterone was detected on average 27.6 days after calving. In first calvers (31.4 +/- 10.2) and in multiparous cows in the winter period (26.9 +/- 9.4) the onset of ovarian activity was delayed compared to start of ovarian activity in the summer period. In the first cycle only 28% of the cows had a normal luteal phase (12-17 days), 36% of the cows had a shortened luteal phase (less than 6 days), and 24% of the cows had a short luteal phase (6-11 days). In 12% of the cows the luteal phase was longer than 17 days. In the second cycle 56% of the cows had a normal luteal phase while 17% had a shortened luteal phase, and 17% had a short luteal phase. Pregnancy rates after first insemination in cows with a short dioestrus (10-25 days) were higher than in cows with a prolonged dioestrus (26-50 days). On the basis of these result it might be expected that postponing the first insemination until the second or even the third cycle in high-yielding cows will have only a marginal effect on the number of open days and a large effect on the number of inseminations per pregnancy.     

Regulation of mRNA encoding low density lipoprotein receptor and high density lipoprotein-binding protein in ovine corpora lutea

Three experiments were conducted to examine the regulation of steady-state concentrations of mRNA encoding ovine low density lipoprotein receptor (LDL-R) and high density lipoprotein-binding protein (HBP) in corpora lutea. In Experiment 1, corpora lutea were collected from ewes on Days 3, 6, 9, 12 and 15 (Day 0, oestrus) of the oestrous cycle. Enriched preparations of small and large steroidogenic luteal cells were also obtained on Days 6, 9, 12 and 15 of the oestrous cycle. In Experiment 2, 16 ewes were hypophysectomized on Day 5 of the oestrous cycle and received saline, luteinizing hormone (LH), growth hormone (GH) or a combination of LH+GH until collection of luteal tissue on Day 12 of the oestrous cycle. Corpora lutea were also collected from pituitary-intact control ewes on Day 5 and Day 12 of the oestrous cycle. In Experiment 3, 13 ewes on Day 11 or Day 12 of the oestrous cycle were administered prostaglandin F2 alpha (PGF2 alpha) and corpora lutea were collected 4 h, 12 h and 24 h later. Corpora lutea were also collected from 4 non-injected and 4 saline-injected (at 24 h) ewes. Results demonstrated that concentrations of mRNA encoding LDL-R did not differ throughout the oestrous cycle. Luteal tissue collected on Day 3 of the oestrous cycle had higher concentrations of mRNA encoding HBP than luteal tissue collected on any other day of the oestrous cycle. Hypophysectomy increased concentrations of mRNA encoding LDL-R but had no effect on concentrations of mRNA encoding HBP. Twelve hours following PGF2 alpha injection concentrations of mRNA encoding LDL-R were decreased but concentrations of mRNA encoding HBP were increased. Concentrations of both LDL-R and HBP mRNA were decreased 24 h following injection of PGF2 alpha. Thus, long-term positive and acute negative regulation of progesterone secretion from the corpus luteum by luteotrophic and luteolytic hormones was not mediated by changes in steady-state concentrations of mRNA encoding LDL-R or HBP.     

Utility of serum progesterone and prolactin analysis for assessing reproductive status in the Asian elephant (Elephas maximus)

Concentrations of serum progesterone and prolactin were assessed during the perioestrous period and throughout gestation in the Asian elephant (Elephas maximus) as a means of generating information of potential use to managers. In > 95% of perioestrous periods (n=35), behavioural oestrus (as determined by bull interest, mounting and/or breeding) coincided with the onset of increased serum progesterone concentrations at the beginning of the luteal phase and continued through Day 7 (Day 1 = first significant serum progesterone rise). Within individuals, 1- to 2-day transient decreases (P < 0.05) in serum progesterone occurred between Days 2 and 9. Notably, no sexual behaviour was observed in any female after this transient fall in progesterone. Prolactin concentrations fluctuated randomly throughout the perioestrous period, with no clear pattern. During the study, four females conceived (one conceived twice), and two delivered three viable offspring. Serum progesterone was elevated above baseline throughout gestation, and then declined precipitously 2-3 days before parturition. Serum prolactin concentrations were significantly elevated above baseline (P < 0.05) after 5-6 months of gestation and remained high until after parturition. This study confirms that serum progesterone and prolactin analyses are useful tools for monitoring the reproductive status of Asian elephant females. Specifically, the transition from low to high progesterone secretion during the late interluteal/early luteal phase is predictive of oestrus and can be used to coordinate breeding efforts. Pregnancy can be confirmed by elevated serum prolactin after 6 months postbreeding, whereas the late gestational decrease in progesterone is predictive of impending parturition.     

The activity of catechol-O-methyltransferase and monoamine oxidase in the uterine artery of pigs during the oestrous cycle

Activities of catechol-O-methyltransferase (COMT), total monoamine oxidase (MAO) and both types of MAO-A and MAO-B activities were examined in uterine artery on the 0-2nd, 13-14th and 16-18th days of the oestrous cycle in pigs. It was shown that activity of COMT was the lowest on the 0-2nd day, while on the 16-18th day of the oestrous cycle it increased by 52.4% (p < 0.05). Total activity of MAO was the highest at periovulatory phase, whereas on days 13-14 and 16-18 of oestrous cycle is was lower by 83.5% (p < 0.01) and 58.1% (p < 0.01) compared with its activity at periovulatory phase, and was higher on day 16-18 by 153.3% (p < 0.01) in relation to the luteal phase (13-14th day). MAO-A activity was 31.3% (p < 0.01) and MAO-B 62.5% (p < 0.05) of the total activity of MAO. Their activities were also highest at periovulatory phase, then decreased by 86.8-87.4% (p < 0.01) on 13-14th day and by 54.8-57.5% (p < 0.01) or 16-18th day of oestrous cycle. Activities of MAO-A and MAO-B were higher by 223.0-258.2% (p < 0.01) on 16-18th day in relation to the luteal phase (13-14th day). On that base we suppose that variations of COMT and MAO activities can significantly change the catecholamines content in the blood vessels of reproductive organs of pigs.     

Bovine plasma oestrogens, progesterone and glucocorticoids during dexamethasone induced parturition

Plasma samples were collected from jugular, uterine and utero-ovarian veins during glucocorticoid induced parturition. Plasma oestrogens, corticosteroids and progesterone were determined by competitive protein binding methods. Corticosteroids and progesterone began to decline within 8 to 10 h following DXMS treatment. Corticoids were only temporarily suppressed, while progesterone fell to minimum levels and remained low through calving. At this stage of gestation (270 days) peripheral plasma progesterone was primarily of ovarian origin. Pre-treatment with HCG appeared to support progesterone production by the CL despite DXMS treatment in 2 of 6 cows. These 2 cows failed to calve within the expected 96 h after DXMS. Plasma oestrogens did not show significant increased until 24 h after DXMS treatment. Cows which responded to DXMS treatment (calved) had significantly higher oestrogen levels than those which did not respond. It was concluded that oestrogens probably play a permissive rather than an initiating role in parturition.     

In vivo phosphorylation of phosphofructokinase from the bivalve mollusk Mytilus galloprovincialis

The objective of this study was to determine whether progesterone prevents the stimulatory effects of oestradiol on GnRH receptor gene expression. In Expt 1, ewes were treated during the luteal phase (days 10-12 of the oestrous cycle) with either one or five subcutaneous implants containing oestradiol (n = 6 per group). Control ewes received no treatment (n = 6). Anterior pituitary glands were collected 16 h after treatment with oestradiol. Steady-state amounts of GnRH receptor mRNA were similar among all three treatment groups despite increased circulating concentrations of oestradiol in implanted ewes at the time of pituitary collection (4.3 +/- 0.6 and 24.7 +/- 2.6 pg ml-1 in ewes treated with one or five implants, respectively, compared with 0.5 pg ml-1 in controls). Experiment 2 was designed to determine whether progesterone was the ovarian factor preventing the stimulatory effects of oestradiol on expression of the GnRH receptor gene in Expt 1. Twenty-five ewes were ovariectomized on day 6 or day 7 of the oestrous cycle and assigned to one of five treatment groups (n = 5 per group). Control ewes received no further treatment. Endogenous luteal phase concentrations of progesterone were replaced in three groups of ewes at the time of ovariectomy via intravaginal implants. Three days after ovariectomy, one group of progesterone-treated ewes received one oestradiol implant, while another group of progesterone-treated ewes received five oestradiol implants. An additional group was treated with five oestradiol implants only, and anterior pituitary glands were collected from all ewes 16 h later. Compared with untreated ovariectomized ewes, treatment with progesterone alone did not affect amounts of GnRH receptor mRNA. In ewes treated with progesterone and either one or five oestradiol implants, steady-state amounts of GnRH receptor mRNA were increased twofold (P < 0.01). Treatment with oestradiol in the absence of progesterone increased amounts of GnRH receptor mRNA threefold (P < 0.001). These results provide evidence that the stimulatory effects of oestradiol on the expression of the GnRH receptor gene are prevented during the natural luteal phase in ewes. However, progesterone does not appear to act independently to mediate this effect.     

Recombinant human inhibin-A administered early in the menstrual cycle alters concurrent pituitary and follicular, plus subsequent luteal, function in rhesus monkeys

Inhibin, a suppressor of pituitary FSH secretion in nonprimate species, may also act in the ovary to regulate follicular development. To examine whether inhibin has similar actions in primates, female rhesus monkeys (n = 3/treatment), exhibiting regular menstrual cycles, received sc injections of either vehicle or 60 micrograms/kg recombinant human inhibin-A at 0800 and 1600 h for 5 days beginning at menses. The vehicle-treated monkeys displayed menstrual cycles of normal length, with the follicular (11.3 +/- 2.5 days, mean +/- SE) and luteal (16.3 +/- 2.5 days) phases demarcated by midcycle peaks in serum estradiol (E) and bioactive LH. After the first inhibin injection, levels of immunoreactive inhibin A peaked at 10 ng/mL within 1 h and returned to baseline (< 0.1 ng/mL) before the second injection 8 h later. Although serum E and LH did not change, bioactive FSH decreased (to 66% of pretreatment levels, P < 0.05) within 8 h. Within 1 day, circulating bioactive FSH was less (P < 0.05) in inhibin-treated monkeys, compared with controls. By 2-3 days, serum E levels were also markedly (P < 0.05) reduced in inhibin-treated animals, whereas bioactive LH rose 3-fold (P < 0.05). After inhibin treatment, the midcycle rises in serum E and LH were delayed; hence, the follicular phase was prolonged (15.0 +/- 2.6 days, P < 0.05), compared with controls. Although the patterns and levels of serum LH circulating during the subsequent luteal phase seemed comparable in both groups, mean progesterone levels were suppressed to 2-3 ng/mL (P < 0.05) during the midluteal phase in inhibin-treated monkeys. However, the length of the luteal phase in inhibin-treated cycles (13.0 +/- 2.6 days) was not significantly altered. We conclude that exogenous inhibin rapidly diminishes pituitary FSH secretion in female monkeys during the early follicular phase of the menstrual cycle. This action, and/or other actions directly on the ovary, leads to subsequent effects on follicular steroidogenesis and pituitary LH secretion that culminate in an aberrant ovarian cycle characterized by an insufficient luteal phase. The study identifies, for the first time, possible activities and roles of inhibin during the ovarian cycle in primates.     

Oestrogen in human pregnancy faeces

The oestrogen content of two 24 h pools of pregnancy faeces, obtained from 2 normal women in the 33rd-37th week og gestation, was studied. The qualitative analyses were made by gas chromatography - mass spectrometry and the quantitative analyses by mass fragmentography. The presence of the following oestrogens in pregnancy faeces was established: Oestriol, oestrone, oestradiol-17 beta, 16-epioestriol, 17-epioestriol, 16 alpha-hydroxyoestrone, 16-oxo-oestradiol-17 beta, 15 alpha-hydroxyoestrone and 15 alpha-hydroxyoestradiol-17 beta. In addition, mass fragmentographic evidence was obtained for the presence of 16 beta-hydroxyoestrone, 2-methoxyoestrone and oestradiol-17 alpha. The total oestrogen excretion determined in the two pools was 786 and 1300 mug per 24 h. Unconjugated oestrogens accounted for 97.8 and 98.6% of these amounts, respectively. Oestriol, oestradiol-17 beta, 15 alpha-hydroxyoestradiol-17 beta, 16-epioestriol and oestrone, in that order, were quantitatively the most significant of the oestrogens determined. The remarkably high levels of oestradiol-17 beta fround in faeces show, that in pregnancy, this mode of excretion is as important as urine for the elimination of this biologically active steroid. It is suggested that some of the oestradiol may have b-en formed through bacterial enzyme action from other oestrogens or neutral steroids. Only trace amounts of ring D alpha-ketolic oestrogens were found in faeces. This is in marked contrast to the considerable amounts of these steroids found in pregnancy bile and urine.     

A longitudinal study of maternal serum inhibin-A, inhibin-B, activin-A, activin-AB, pro-alphaC and follistatin during pregnancy

Maternal serum concentrations of inhibin-A, inhibin-B, activin-A, activin-AB, pro-alphaC-related inhibin forms, total follistatin, steroids and gonadotrophins were measured longitudinally in six normal singleton pregnancies. Maternal venous blood was collected randomly during a spontaneous follicular phase prior to donor insemination, at 5, 7, 9, 11, 16, 20, 24, 28, 32 and 36 weeks after the first missed menses and in the early puerperium. Steroid and gonadotrophin profiles conformed to previous reports. While at week 5 of gestation inhibin-A, activin-A and follistatin concentrations were similar to those at the follicular phase, all three increased progressively (P < 0.001) to maximal concentrations in week 36: approximately 48-fold (3740 +/- 1349 ng inhibin-A/ml), approximately 22-fold (6109 +/- 1443 ng activin-A/ml) and approximately 10-fold (3563 +/- 418 ng follistatin/ml) higher. Pro-alphaC concentrations reached a maximum in weeks 5 (approximately 5-fold, P < 0.001) and 36 (1027 +/- 174 pg/ml, P < 0.01). Inhibin-B (71 +/- 23 pg/ml prior to pregnancy) was undetectable (<12 pg/ml) between week 5-16 of gestation but increased slightly in the third trimester (26 +/- 7 pg/ml in week 36). Activin-AB was undetectable throughout pregnancy. Post-partum concentrations of inhibin-A (41 +/- 12 ng/ml), inhibin-B (<12 pg/ml), activin-A (950 +/- 149 pg/ml), pro-alphaC (128 +/- 22 pg/ml) and follistatin (990 +/- 79 ng/ml) were substantially lower than at week 36 of gestation. The activin-A:follistatin ratio increased from 0.5 in week 5 to 1.8 in week 36, suggesting that more free activin-A is available in the maternal circulation during late pregnancy.     

Monoamine oxidase activity in the uterine and mesenteric arteries, vessels of ovarian pedicle and myometrium of pigs during the oestrous cycle

Activities of total monoamine oxidase (MAO-T) and of both subtypes: MAO-A and MAO-B were examined in the uterine artery, vessels of ovarian pedicle (subovarian veno-arterial network), mesenteric artery and myometrium on the 0-2nd, 13-14th and 16-18th days of the oestrous cycle of pigs. It was found that in the uterine artery MAO-T, MAO-A and MAO-B activities were the highest on the 0-2nd days (100%); on day 13-14 they were lower by 85.4, 84.8 and 87.4% (p < 0.01), respectively, and on days 16-18 they were lower by 52.4, 57.5 and by 54.1% (p < 0.01) in comparison with the value on the 0-2nd days of ovarian cycle. The activities of MAO-T, MAO-A and MAO-B in the vessels of ovarian pedicle on the 0-2nd days were the lowest, whereas on days 13-14 they were the highest (p < 0.01), MAO-T by 445.3%, MAO-A by 315.6%, MAO-B by 344.7%, and they were higher on days 16-18 by 133.8% (p < 0.01), 85.9 (p < 0.05) and by 281.5% (p < 0.01) in comparison with the value on the 0-2nd day of ovarian cycle, respectively. The activities of examined enzymes in mesenteric artery were higher on 0-2nd and 16-18th days, whereas MAO-T and MAO-B they were lower by 39.0% and MAO-A by 22.3% (p < 0.05) on 13-14th days of oestrous cycle. In myometrium the activities of MAO-T, MAO-A and MAO-B on the 0-2nd days (100%) were the lowest, whereas on days 13-14 the activities were the highest (p < 0.01), MAO-T by 326.3%, MAO-A by 363.3% and MAO-B by 364.1%, and on days 16-18 the MAO-T and MAO-B activities were also higher by 99.4% and 150.7% (p < 0.01) respectively, whereas MAO-A activity was similar to the value on the 0-2nd days. The high MAO activity in the vessels of pig ovarian pedicle may be a significant factor in the ovarian vasotone decrease and increase in the ovarian blood flow during the luteal phase of ovarian cycle.     

Prolonged inhibition of normal ovarian cycles in the rat and cynomolgus monkeys following a single s.c. injection of danazol

In castrated male rats, a single s.c. injection of danazol has been shown to result in an inordinately prolonged inhibition of serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) concentrations. In the present study, we have examined whether the same and similar routes of administration suppresses ovarian function in normally cycling rats and cynomolgus monkeys. Normally cycling female rats received danazol as a single administration either orally, i.m. or s.c. and a separate group also received danazol in silastic capsules. The duration of the dioestrous interval until the next oestrous smear was followed daily and cycle lengths were compared with vehicle-treated groups. Six normally cycling cynomolgus monkeys were followed by daily observation and blood sampling at 2-3 day intervals. After one normal cycle, danazol (200 mg/kg) was administered as a single s.c. injection. Monkeys were followed until the next menses and one cycle thereafter and blood samples were assayed for oestradiol, progesterone and bioactive LH. Oestrous cycle length in vehicle-treated control rats was 4.7 days. A single administration of danazol s.c. at the higher dose prolonged the dioestrous interval to 31.3 days (P <0.001) and a similar prolongation was observed with this high dose when administered i.m. (27.7 days; P <0.001). In normally cycling monkeys, the menstrual cycle length was 30.2 days, but following a single danazol administration, the mean duration to the next menses was prolonged to 117.5 days (P <0.001). In five out of six monkeys, there was a decrease in LH and an absence of normal oestradiol and progesterone patterns. After this prolonged hiatus, a subsequent menstrual cycle was normal in length and endocrine pattern. A single s.c. administration of danazol resulted in a prolonged suppression of ovarian cyclicity in both normally cycling rats and cynomolgus monkeys.     

Ultrastructural localization of relaxin in the corpus luteum of the pregnant and early lactating tammar wallaby, Macropus eugenii

Electron-microscope immunocytochemistry was used to determine the subcellular distribution and presence of immunoreactive relaxin throughout pregnancy and early lactation in the corpus luteum of a marsupial, the tammar wallaby. Membrane-bound, electron-dense granules were a prominent feature of the luteal cell cytoplasm. The highest numbers of granules were observed between days 20 and 24 of the 26-day gestation, with a rapid clearance immediately after birth. Relaxin immunogold particles were present only in small, electron-dense granules (200-350 nm in diameter), with no particles observed in larger granules (>400 nm diameter), nuclei or mitochondria. Relaxin immunoreactivity was low throughout early and mid pregnancy but increased markedly between days 21 and 22 and remained high over the last 4 days of pregnancy. The number of granules containing relaxin immunogold particles and the density of immunostaining were both reduced on the day of expected births (day 26). Our data demonstrate that electron-dense granules in the luteal cell cytoplasm of a pregnant marsupial contain relaxin. The peptide is produced in greatest amounts at the end of pregnancy, consistent with a role in parturition.     

alpha-Tocopherol concentrations in plasma but not in lipoproteins fluctuate during the menstrual cycle in healthy premenopausal women

Because premenopausal women experience cyclic fluctuations of plasma carotenoids and their lipoprotein carriers, it was hypothesized that plasma alpha-tocopherol (A-T) fluctuates by phase of the menstrual cycle. Twelve free-living women, with a confirmed ovulatory cycle, were given a controlled diet for two consecutive menstrual cycles. Blood was drawn during the menses, early follicular, late follicular and luteal phases to simultaneously measure serum hormones, plasma lipoproteins and A-T concentrations, and A-T distribution in the lipoprotein fractions. Plasma A-T concentrations were significantly lower during menses than during the luteal phase by approximately 12% in each controlled diet cycle (P < 0.001). Adjustment for serum cholesterol and triglyceride concentrations did not alter these findings. The distributions of A-T in lipoprotein cholesterol fractions were not significantly different by menstrual phase. From 61 to 62% of A-T was concentrated in the LDL fraction, with another 9-14% in HDL2, 17-22% in HDL3 and the remaining 6-8% in VLDL+ IDL. There were no significant differences in lipoprotein cholesterol fractions by menstrual phase, except for a significant increase (P = 0.03) in HDL2 cholesterol from the early follicular to the late follicular phase. Spearman rank correlations from data during the second controlled diet month showed A-T in HDL2 in the late follicular phase was positively correlated with HDL cholesterol in the early follicular (r = 0.88), late follicular (r = 0.86) and luteal phases (r = 0.86) and with luteal apolipoprotein (ApoA-1) level (r = 0.90), and luteal HDL2 cholesterol (r = 0.83). A-T in HDL3 in the early follicular phase was negatively correlated with HDL2 cholesterol (r = -0.96) and ApoA-1 (r = -0.85), whereas luteal A-T in HDL3 was correlated with luteal HDL3 cholesterol (r = -0.79). Late follicular A-T in VLDL was positively correlated with early follicular HDL3 cholesterol and late follicular HDL3 cholesterol (r = 0.83). Fluctuations of A-T concentrations by phase of the menstrual cycle should be taken into consideration in future research concerning premenopausal women and the risk of chronic disease.     

Body condition at parturition and postpartum weight gain influence luteal activity and concentrations of glucose, insulin, and nonesterified fatty acids in plasma of primiparous beef cows

Effects of body condition score (BCS) at parturition and postpartum weight gain on luteal activity and concentrations of glucose, insulin, and NEFA in plasma were evaluated during the breeding season in 242 primiparous beef cows over 3 yr (Y) at three locations (L). At approximately 90 d prepartum, cows were blocked by breed, expected calving date, and BCS and randomly assigned to diets so that cows would calve in BCS of 4, 5, or 6. At calving, cows were blocked by breed, calving date, and BCS and randomly allotted to gain .45 (M) or .90 (H) kg/d, from parturition to the start of breeding (postpartum nutrition; PPN). During the 60-d breeding season, weekly blood samples were obtained from cows, and progesterone, insulin, glucose, and NEFA were quantified. Progesterone concentrations greater than 1 ng/mL for more than 1 wk indicated luteal activity. To determine the possible value of blood constituents as predictors of luteal activity, categorical data analyses were performed. Cows with greater BCS at parturition had greater concentrations of glucose during breeding (P < .07). Similarly, PPN influenced glucose at the beginning of breeding, but the differences were minimal after d 28 (PPN x day; P <.001). Cows with greater BCS at parturition and M-PPN had greater concentrations of insulin during the breeding season (BCS x PPN; P < .02). Cows with a BCS of 6 at parturition had the lowest concentrations of NEFA; however, cows on H-PPN had greater concentrations of NEFA (BCS x PPN; P < .03). Location, BCS, PPN, and day affected luteal activity (P < .002). Location differences in luteal activity were associated with the interval from calving to the start of breeding. In general, a greater percentage of cows with BCS of 5 or 6 at calving had luteal activity by the end of the breeding season. Concentrations of metabolites in blood during breeding were not predictive of luteal activity. We conclude that BCS at parturition and postpartum nutrition influence concentrations of glucose, insulin, and NEFA in blood and the onset of luteal activity in primiparous beef cows.     

Stress and the menstrual cycle: relevance of cycle quality in the short- and long-term response to a 5-day endotoxin challenge during the follicular phase in the rhesus monkey

The notion that stress activates central and peripheral pathways to inhibit the menstrual cycle is well accepted, but the initial processes through which this occurs have not been investigated. This study uses a relevant nonhuman primate model to document the cyclic endocrine effects imposed by a moderate short-term stress episode in the follicular phase. The stress paradigm is a 5-day inflammatory/immune-like challenge produced by the administration of bacterial endotoxin [lipopolysaccharide (LPS)], which, through the release of endogenous cytokines and other mediators, induces a physiopathological response similar to a bacterial infection. LPS was administered iv twice daily for 5 days starting on days 2-8 of the follicular phase. The stress challenge resulted in a significant lengthening of the follicular phase in all monkeys. Two distinct groups were observed. In group 1 (n = 5), the mean (+/- SE) length of the follicular phase in the LPS-treated cycle was significantly increased, from 10.2 +/- 0.2 in control cycle 2 to 30.8 +/- 4.3 days (except in one monkey that had a 4-month amenorrheic interval). In group 2 (n = 5), the length of the follicular phase significantly increased but not to exceed the duration of the LPS treatment (9.7 +/- 1.1 vs. 13.6 +/- 1.2). Estradiol concentrations decreased significantly after LPS in group 1 (34.8 +/- 5.5 vs. 16.2 +/- 6.5 pg/mL) and remained suppressed after the challenge. In group 2, estradiol levels remained stationary throughout the 5-day LPS treatment (26.0 +/- 6.5 vs. 25.6 +/- 3.9). Compared with control values at a similar stage of the follicular phase, most LH and FSH values during LPS treatment were higher than controls. Estradiol and gonadotropin surges were delayed by LPS treatment for a varying length of time according to each grp. Significant differences in integrated luteal progesterone concentrations characterized control cycles of groups 1 and 2 (group 1: 36.5 +/- 1.5, group 2: 47.5 +/- 2.6). In group 1, there were no further effects of LPS on luteal progesterone during the treatment and two post-LPS cycles. In contrast, in group 2, integrated luteal progesterone concentrations were significantly decreased in post-LPS cycle 1 (to 36.0 +/- 4.4). Cortisol significantly increased at hour 3 after each morning LPS injection but the amplitude of the response decreased over the 5-day period. Progesterone increased significantly by hour 3 after the first LPS injection but remained unchanged after subsequent LPS administration. Our data demonstrate that a 5-day inflammatory-like episode during the follicular phase can delay folliculogenesis and that damage to this process is intensified in individuals who already demonstrate a subtle cyclic degradation, in the form of decreased progesterone secretion in the luteal phases preceding the stress episode. Long-term endocrine effects, in the form of decreased luteal secretory activity in the first poststress cycle, are observed in normally cycling individuals, suggesting that inadequacy of the luteal phase may represent the first stage in the damage that a stress episode can inflict upon the normal menstrual cycle.     

Prostaglandin F and progesterone secretion by porcine endometrium and corpus luteum in vitro

Slices of porcine endometrium and corpus luteum tissue obtained from mature sows throughout the luteal phase of the oestrous cycle were incubated in culture medium which was analysed at regular intervals over a period of 8 hours for prostaglandin F and progesterone. Prostaglandin F secretion was greatest by endometrium obtained during the mid III to late I luteal stage of the cycle and the increased levels secreted by this tissue were paralleled by high levels of secretion from corpus luteum tissue. The addition of indomethacin (10 mug/ml) to the culture medium completely abolished prostaglandin F secretion by both endometrium and luteal tissue indicating that the high levels of the prostaglandin were due to synthesis. Progesterone secretion by the corpus luteum was maximal from early luteal tissue and had declined to considerably lower levels by late stage tissue when prostaglandin secretion was greatest. The possible physiological significance of luteal prostaglandin F secretion is discussed.