Reproductive wastage in the androstenedione-immune ewe

An experiment was carried out using 320 adult Merino ewes to examine the effects of immunization against an androstenedione human serum albumin conjugate (Fecundin) on ovulation rate, fertilization rate and embryo viability at days 2, 9 and 13-14 after fertilization. The ovulation rate of immunized ewes (2.19 +/- 0.06) was greater (P < 0.001) than that of control ewes (1.43 +/- 0.04). The recovery rate of embryos or of unfertilized oocytes on day 2 was reduced in immunized ewes, but fertilization rate of recovered oocytes was unaffected by immunization. The mean number of normal embryos per ewe pregnant (prolificacy) was higher and the proportion of ewes pregnant (fertility) was lower in immunized than in the control ewes. The distribution of the number of cells per embryo showed no differences in developmental age over the period of sampling, the majority of embryos at this time being at the two- to four-cell stage of development. At day 9 of pregnancy, blastocyst recovery rates were lower in immunized than in control ewes. About 90% of blastocysts recovered were developing normally in control ewes compared with 64% in immunized ewes. The majority of blastocysts recovered on day 9 had hatched from the zona pellucida prior to recovery (mean values were 94.2% and 87.8% for control and immunized groups, respectively). In control ewes single blastocysts were larger than twin blastocysts, but for the immunized ewes this difference was not significant. Both single blastocysts (P < 0.01) and twin blastocysts (P < 0.05) from immunized ewes were smaller than those from control ewes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of oestradiol on ovarian oxytocin secretion rate and luteolysis in the ewe after ovarian auto-transplantation

The release of ovarian oxytocin and uterine prostaglandin (PG)F2alpha in response to an oestradiol stimulus was investigated. On Day 15 post-oestrus, ten ewes with ovarian auto-transplants (n=5 per group) received an intra-muscular injection of either oestradiol benzoate (50 microg) or vehicle. Blood samples were collected from the ovarian and jugular veins at 30 and 0 min before, and at 15-min intervals up to 540 min after, injection. The secretion rate of ovarian progesterone remained elevated in four of five treated ewes and in all control ewes, indicating the presence of a functional corpus luteum. Peripheral oestradiol concentrations were significantly (P < 0.001) higher in treated than in control ewes. The number of ewes that released pulses of ovarian oxytocin > or =240 min following oestradiol benzoate injection was significantly (P < 0 05) greater than that in control ewes. Mean amplitude and area under both ovarian-vein oxytocin and jugular-vein 15 keto-13,14 dihydro prostaglandin F2alpha (PGFM) pulses were significantly increased in the treated ewes. These findings demonstrate that the administration of exogenous oestrogen provides a positive stimulus for the release of ovarian oxytocin and uterine PGF2alpha in the ovarian auto-transplanted ewe.     

Changes in the secretion of ovarian steroid and pituitary luteinizing hormone in the peri-ovulatory period in the ewe: the effect of progesterone

The secretion rates of oestradiol, androstenedione and progesterone and the peripheral plasma concentration of LH were measured in 12 ewes with ovarian autotransplants before and after luteal regression induced by a single intramuscular injection of a synthetic prostaglandin (PG) analogue, 16-aryloxyprostaglandin F 2alpha (I.C.I. 80996). Luteal regression was followed by a fourfold rise in the basal concentration of LH and increased secretion of oestradiol. In five out of six ewes there was a discharge of LH with the peak occurring 36--78 h after the injection of the PG analogue. The secretion of oestradiol declined from 3-68 +/- 1-08 to 0-33 +/- 0-6 (S.E.M.) ng/min in the 24 h following the LH peak (P less than 0-001). In the remaining six ewes in which progesterone was implanted subcutaneously 24 h after the injection of PG analogue, follicular development was suppressed as indicated by the low secretion of oestradiol and androstenedione. The basal concentration of LH fell to values similar to those observed during the luteal phase after the implant of progesterone. The secretion of androstenedione followed a similar pattern to that of oestradiol in those ewes which showed presumptive evidence of ovulation. These results suggest that progesterone reinforces the negative feedback effects of oestrogen in the ewe.     

Uterine contractions at the time of embryo transfer alter pregnancy rates after in-vitro fertilization

To investigate the possible consequences of uterine contractions (UC) as visualized by ultrasound (US) on in-vitro fertilization (IVF)-embryo transfer outcome, we studied prospectively 209 infertile women undergoing 220 cycles of controlled ovarian stimulation. Inclusion criteria were age < or = 38 years, a morphologically normal uterus, and at least three good quality embryos transferred. Just before embryo transfer, women underwent 5 min digital recordings of the uterus using US image analysis software for UC assessment. Plasma progesterone and oestradiol concentrations were measured. Four groups were defined according to UC frequency: < or = 3.0 (n = 53), 3.1-4.0 (n = 50), 4.1-5.0 (n = 43), and > 5.0 (n = 74) UC/min respectively. Patients, controlled ovarian hyperstimulation and embryology characteristics were comparable in all groups. A stepwise decrease in clinical and ongoing pregnancy rates as well as in implantation rates occurred from the lowest to the highest UC frequency groups (53, 36, 21; 46, 32, 20; 23, 19, 10; and 14, 11, 4%; P < 0.001). Plasma progesterone and UC frequency were negatively correlated (r = -0.34, P < 0.001). Direction of UC did not affect embryo transfer outcome. As this study was controlled strictly for confounding variables and UC were assessed objectively by a computerized system, its results indicate that high frequency UC on the day of embryo transfer hinder IVF-embryo transfer outcome, possibly by expelling embryos out of the uterine cavity. The negative correlation between UC frequency and progesterone concentrations supports the uterine relaxing properties of progesterone.     

A method for endoscopic embryo collection and transfer in the rabbit

Thirty-two rabbits were used for endoscopic embryo collection and transfer. For embryo collection midventral laparoscopy and transcervical endoscopy were combined for orthograd flushing of the oviducts and uterine horns. Transfer was performed transcervically under laparoscopic control. The mean number of corpora lutea counted in nine donors was 13.3 +/- 8.4. A total of 72 morulae/blastocysts were obtained. No embryos were recovered when only the uterine horns were flushed. 291 embryos were transcervically transferred to 23 recipients which resulted in 10 pregnant animals at day 12. Two of three slaughtered recipients showed together 4 implantation sites. Four animals delivered 12 pups.     

Regulation of oxytocin, oestradiol and progesterone receptor concentrations in different uterine regions by oestradiol, progesterone and oxytocin in ovariectomized ewes

The regulation of oxytocin, oestradiol and progesterone receptors in different uterine cell types was studied in ovariectomized ewes. Animals were pretreated with a progestogen sponge for 10 days followed by 2 days of high-dose oestradiol to simulate oestrus. They then received either low-dose oestradiol (Group E), low-dose oestradiol plus progesterone (Group P) or low-dose oestradiol, progesterone and oxytocin (via osmotic minipump; Group OT). Animals (three to six per time-point) were killed following ovariectomy (Group OVX), at oestrus (Group O) or following 8, 10, 12 or 14 days of E, P or OT treatment. In a final group, oxytocin was withdrawn on day 12 and ewes were killed on day 14 (Group OTW). Oxytocin receptor concentrations and localization in the endometrium and myometrium were measured by radioreceptor assay, in situ hybridization and autoradiography with the iodinated oxytocin receptor antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]-vasotocin. Oestradiol and progesterone receptors were localized by immunocytochemistry. Oxytocin receptors were present in the luminal epithelium and superficial glands of ovariectomized ewes. In Group O, endometrial oxytocin receptor concentrations were high (1346 +/- 379 fmol [3H]oxytocin bound mg protein-1) and receptors were also located in the deep glands and caruncular stroma in a pattern resembling that found at natural oestrus. Continuing low-dose oestradiol was unable to sustain high endometrial oxytocin receptor concentrations with values decreasing significantly to 140 +/- 20 fmol mg protein-1 (P < 0.01), localized to the luminal epithelium and caruncular stroma but not the glands. Progesterone treatment initially abolished all oxytocin receptors with none present on days 8 or 10. They reappeared in the luminal epithelium only between days 12 and 14 to give an overall concentration of 306 +/- 50 fmol mg protein-1. Oxytocin treatment caused a small increase in oxytocin receptor concentration in the luminal epithelium on days 8 and 10 (20 +/- 4 in Group P and 107 +/- 35 fmol mg protein-1 in Group OT, P < 0.01) but the rise on day 14 was not affected (267 +/- 82 in Group OT and 411 +/- 120 fmol mg protein-1 in Group OTW). In contrast, oestradiol treatment was able to sustain myometrial oxytocin receptors (635 +/- 277 fmol mg protein-1 in Group O and 255 +/- 36 in Group E) and there was no increase over time in Groups P, OT and OTW with values of 61 +/- 18, 88 +/- 53 and 114 +/- 76 fmol mg protein-1 respectively (combined values for days 8-14). Oestradiol receptor concentrations were high in all uterine regions in Group O. This pattern and concentration was maintained in Group E. In all progesterone-treated ewes, oestradiol receptor concentrations were lower in all regions at all time-points. The only time-related change occurred in the luminal epithelium in which oestradiol receptors were undetectable on day 8 but developed by day 10 of progesterone treatment. Progesterone receptors were present at moderate concentrations in the deep glands, caruncular stroma, deep stroma and myometrium in Group O. Oestradiol increased progesterone receptors in the luminal epithelium, superficial glands, deep stroma and myometrium. Progesterone caused the loss of its own receptor from the luminal epithelium and superficial glands and decreased its receptor concentration in the deep stroma and myometrium at all time-points. There was a time-related loss of progesterone receptors from the deep glands of progesterone-treated ewes between days 8 and 14. These results show differences in the regulation of receptors between uterine regions. In particular loss of the negative inhibition by progesterone on the oxytocin receptor by day 14 occurred only in the luminal epithelium, but is unlikely to be a direct effect of progesterone as no progesterone receptors were present on luminal epithelial cells between days 8 and 14.     

Effects of exogenous recombinant ovine interferon tau on circulating concentrations of progesterone, cortisol, luteinizing hormone, and antiviral activity; interestrous interval; rectal temperature; and uterine response to oxytocin in cyclic ewes

Interferon tau (IFN tau) is the conceptus-produced antiluteolytic signal in ruminants. Three experiments examined the effects of s.c. administration of recombinant ovine (ro)IFN tau on interestrous interval (IEI), oxytocin (OT)-induced uterine prostaglandin F2alpha metabolite (PGFM) production, rectal temperature (RT), respiration rate (RR), and plasma concentrations of progesterone, cortisol, LH, and antiviral activity (AVA) in plasma and uterine flushings. In experiment I, 20 ewes were treated s.c. with either 0, 1, 2, or 4 mg/day roIFN tau (0.7 x 10(8) U/mg; 5 ewes/dosage) from Days 11 to 15 of the estrous cycle (estrus = Day 0) and were challenged with OT (30 IU) on Day 15. Jugular blood samples were collected at -10, 0, 10, 20, 30, 40, 50, and 60 min relative to the OT challenge and assayed for PGFM. Recombinant oIFN tau increased IEI (16.7, 18.7, and 22.6 +/- 0.6 days for 0, 2, and 4 mg roIFN tau, respectively, p < 0.01). Recombinant oIFN tau did not affect peak PGFM response to OT (2309 +/- 172 pg/ml; p > 0.1). However, the 4 mg/day dosage delayed the time to peak PGFM (32.4 vs. 47.5 +/- 3.4 min; p < 0.01, 0 vs. 4 mg) and resulted in approximately 200% higher concentrations of PGFM at 60 min post-OT (0 vs. 4 mg/day, p < 0.07). Experiment II was similar to experiment I, except that only the 0- and 4-mg/day dosages of roIFN tau were administered. Ewes were hysterectomized on Day 16, and assay of uterine flushes detected no AVA from ewes treated with either 0 or 4 mg/day roIFN tau. In experiment III, 20 ewes were treated s.c. with either 0, 2, 4, or 6 mg roIFN tau on Day 12. Blood samples, RT, and RR were obtained at frequent intervals for 24 h, and plasma was assayed for progesterone, cortisol, LH, and AVA. Plasma AVA, which increased in a dose-dependent manner, was detectable within 60 min and remained elevated at 24 h compared to control values. RT (elevated 0.5-1.0 degrees C), RR, and cortisol increased in response to all dosages of roIFN tau, with peak values occurring 150-180 min postinjection. For all dosages of roIFN tau, plasma progesterone declined from 120 to 360 min posttreatment and then returned to pretreatment values by 24 h (p < 0.01) as compared to controls. Overall, exogenous roIFN tau altered uterine PGFM response to OT from a pulse to a gradual and sustained elevation and extended IEI with only a transient decline in progesterone and mild hyperthermia, effects that are not expected to compromise pregnancy.     

Long-term ovarian function in sheep after ovariectomy and transplantation of autografts stored at -196 C

We have previously demonstrated that ovarian function and fertility can be preserved in sheep after castration by autotransplantation of cryopreserved strips of ovarian cortex. In the current experiments we have investigated the long term survival of such grafts by detailed measurements of ovarian function for a period of nearly 2 yr after autotransplantation. After ovariectomy and transplantation of frozen/thawed grafts, the concentrations of FSH and LH rose to castrate levels for about 14 weeks before falling gradually to reach near-normal levels at about 60 weeks. In the breeding season from October 1994 to March 1995, all ewes had 5-10 estrous cycles that were similar in length to those in the 4 control ewes. Luteal function as indicated by the progesterone concentration was identical before and 11 months after transplantation. In contrast, the basal concentrations of FSH and LH were persistently raised throughout the luteal phase, but showed a normal decline during the follicular phase. The concentration of inhibin A in ovarian venous plasma measured at the end of the experiment 22 months after transplantation was significantly lower than that in control ewes (mean +/- SE, 409 +/- 118 vs. 1914 +/- 555 pg/ml; P < 0.004). Transplantation of frozen/thawed ovarian tissue to SCID mice demonstrated that about 28% of primordial follicles survived the procedure. All of the ovaries transplanted into sheep contained large antral follicles and/or cysts, but very few primordial oocytes when recovered at autopsy after 22 months. These results demonstrate that despite a drastic reduction in the total number of primordial follicles, cyclical ovarian function is preserved in sheep after autotransplantation of frozen/thawed ovarian tissue and provide experimental confirmation that such a technique could provide a means of preserving fertility in women undergoing chemo- or radiotherapy for malignant disease.     

Leigh syndrome: clinical features and biochemical and DNA abnormalities

The morphology and number of cells in the trophectoderm (TE) and inner cell mass (ICM) of buffalo blastocysts derived from in vitro fertilization and cultured in the presence or absence of insulin-like growth factor-I (IGF-I) were analyzed by differential fluorochrome staining technique. The total cell number (TCN), TE number, and ICM cell number were significantly higher in blastocysts developed in vitro in the presence of IGF-I as compared to blastocysts developed without IGF-I (P < 0.01). It was observed that the buffalo blastocyst took 5-9 days postfertilization to develop in vitro. In order to correlate the time required for blastocyst development and the allocation of cells to TE and ICM, blastocysts were designated as fast (developing on or before day 7) or slow (developing after day 7). The TCN, TE, and ICM cells of fast-developing blastocysts cultured in the presence of IGF-I were significantly higher than slow-developing blastocysts (P < 0.01). The blastocysts developed on day 6 had a mean total cell number 118.6 +/- 21.4, which significantly decreased to 85.6 +/- 17.4, 62.0 +/- 14.5, and 17.0 +/- 4.0 on days 7, 8, and 9, respectively (P < 0.05). Normal development of buffalo embryo showed that, on average, embryos reached compact morula stage at the earliest between days 4.5-5.5. Blastocysts developed, at the earliest, between days 5.0-6.0, and it took them, on average, 6.5 days to hatch from the zona pellucida. TCN, TE, and ICM increased three times from morula to blastocyst; however, the proportion of ICM to TCN remained the same, in both embryonic stages. TE approximately doubled in hatched blastocysts, as compared to unhatched blastocysts (P < 0.05). However, ICM cells were decreased. The time required for development of parthenogenetic blastocysts was observed to be greater as compared to in vitro fertilized (IVF) blastocysts. The total cell number of parthenogenetic blastocysts was 100.8 +/- 11.3, including 59.2 +/- 8.4 cells of TE and 42.1 +/- 6.9 cells of ICM.     

Progesterone can block transmission of the estradiol-induced signal for luteinizing hormone surge generation during a specific period of time immediately after activation of the gonadotropin-releasing hormone surge-generating system

The preovulatory GnRH/LH surge in the ewe is stimulated by a rise in the circulating estradiol concentration that occurs in conjunction with preovulatory ovarian follicle development. In the presence of high levels of progesterone, such as during the luteal phase of the estrous/menstrual cycle, the stimulatory effects of elevated estradiol on GnRH/LH secretion are blocked. Recent work in the ewe has shown that a relatively short period of estradiol exposure can stimulate a GnRH/LH surge that begins after estrogenic support has been removed. This result suggests that surge generation is characterized by an estradiol-dependent period (during which the signal is read) and an estradiol-independent period (during which a cascade of neuronal events transmits the stimulatory signal to the GnRH neurosecretory system, which releases a surge of GnRH). In this series of studies, we addressed the hypothesis that progesterone can block transmission of the stimulatory estradiol signal after it has been read. Nine ovariectomized ewes were run through repeated artificial estrous cycles by sequential addition and removal of exogenous steroids. In study one, ewes received three treatments in a randomized cross-over design. Exposure to a follicular phase estradiol concentration for 10 h (positive control treatment) stimulated an LH surge in all ewes, as determined in hourly jugular blood samples. Maintenance of luteal phase progesterone concentrations throughout the artificial follicular phase (2 x CIDR-G devices, negative control) blocked the stimulatory effects of a 10-h estradiol signal, and no ewes that received this treatment expressed an LH surge. In the experimental group, exposure to luteal phase levels of progesterone, during the period after the surge generating system had been activated by estradiol, blocked the LH surge in six of nine ewes. This result demonstrates that progesterone can block the surge, even when applied after the surge-generating system has been activated and, therefore, that it inhibits either the transmission of the estradiol signal and/or the release of the GnRH/LH surge. In study 2, we assessed whether sensitivity to the inhibitory effects of progesterone was confined to a specific stage of the transmission of the estradiol signal. Eight ewes were exposed to four treatments, over successive artificial estrous cycles. Positive and negative controls were similar to those described in Study 1, except the duration of the stimulatory estradiol signal was reduced to 8 h. The two experimental groups consisted of an EARLY P (progesterone) treatment, in which progesterone was given from hours 8-13 after estradiol insertion (immediately after estradiol removal), and a LATE P treatment, in which progesterone was given from hours 13-18 (immediately before LH surge secretion). As expected, LH surges were stimulated and blocked, in response to the positive and negative controls, respectively. Whereas the EARLY P treatment blocked the LH surge in seven of eight ewes, the LATE P treatment was only successful in inhibiting a surge in one of eight animals. This result demonstrates that progesterone can block the estradiol-induced surge-generating signal soon after the onset of signal transmission (immediately after estradiol removal) but not during the later stages of signal transmission (at the time of GnRH/LH surge onset).     

The status of child health and child survival and development programs in Turkey

The effects of nutrition on the testis were investigated in groups of five mature Merino rams that were fed either a sub-maintenance (low) diet or a supra-maintenance (high) diet for 69 days. Testosterone, oestradiol and inhibin were measured in blood plasma sampled simultaneously from jugular and testicular veins after an i.v. injection of 200 ng ovine LH kg-1. Plasma concentrations of testosterone, inhibin and oestradiol were higher in testicular than in jugular vein plasma for both diets (P < 0.01). After the LH injection, jugular plasma testosterone increased more rapidly (P < 0.01) in rams fed the high diet than in rams fed the low diet. This was not seen in the testicular vein. Oestradiol concentrations were higher in rams on the high diet than in those on the low diet, in both the testicular (P < 0.0001) and the jugular vein (P < 0.02). Diet did not affect inhibin concentrations. Testes were surgically removed and processed for light microscopy. Testicular mass and seminiferous tubule length and diameter were higher with the high diet than the low diet (P < 0.01). The number of Sertoli cell nuclei per testis was also affected (high diet: 120 +/- 6 x 10(8); low diet: 77 +/- 7 x 10(8); P < 0.001), whereas the proportion of testis occupied by Sertoli cell nuclei was not affected. The number of Leydig cells per testis was not affected by diet, but Leydig cells occupied a greater volume of testis in rams on the high diet than in those on the low diet (P < 0.001). The effects of nutrition on Leydig and Sertoli cells are consistent with changes in the endocrine and exocrine functions of the testis. The finding that Sertoli cell population was altered in adult rams may be explained by the GnRH-independent effects of nutrition.     

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 effect of the oviduct, uterine, and in vitro environments on zona thinning in the mouse embryo

OBJECTIVE: To evaluate the impact of the oviduct, uterine, and in vitro environments on zona pellucida thinning in the mouse embryo. DESIGN: Female mice were stimulated with pregnant mare serum gonadotropin and mated and hCG injection. Unilateral oviduct ligation was performed on day 2 of gestation using the dorsal approach. The mice were divided into equal groups and killed on days 2, 3, 4, 5, and 10 of gestation. In vitro incubated embryos served as controls. Average daily zona thickness measurements were subjected to analysis of variance and paired Student's t-test. SETTING: The laboratory of the assisted reproductive program of Rush University Medical Center. MAIN OUTCOME MEASURE(S): Progressive daily decrease in average zona thickness. RESULT(S): Zona measurements of embryos flushed out of uterine horns, ligated oviducts, and in vitro incubation demonstrated statistically significant decreases in zona thickness, from 9.6 +/- 1.6 microns (day 3) to 6.0 +/- 0.8 microns (day 5), from 11.6 +/- 2.2 microns (day 2) to 6.0 +/- 1.6 microns (day 5), and from 11.1 +/- 2.0 microns (day 2) to 6.0 +/- 1.6 microns (day 5), respectively. There were no differences in average zona thickness for embryos in the same cell stage and same protocol day in all three locations. CONCLUSION(S): Zona thinning seems to be induced primarily by the dividing embryo before implantation. A substantial tubal and uterine contribution to zona thinning was not detected in this mouse embryo model.     

Control of photoperiodic inhibition of luteinizing hormone secretion by dopaminergic and serotonergic systems in ovariectomized Ile-de-France ewes supplemented with oestradiol

The role of dopaminergic and serotonergic systems on LH secretion was investigated in Ile-de-France ewes under different artificial inhibitory photoperiodic regimens. All animals were ovariectomized at the end of the breeding season, chronically treated with an oestradiol implant, and subjected to various changes in daylength for 9 months to inhibit or stimulate their LH secretion. Plasma LH concentration was assessed by taking blood samples twice a week throughout the experiment. The effects of acute intravenous injections of the dopaminergic2 receptor antagonist pimozide (0.08 mg kg-1) and the 5-hydroxytryptamine2 (5HT2) receptor antagonist cyproheptadine (3 mg kg-1) on LH pulsatility were assessed during challenges in four different situations: (1) long days (LD); (2) before short-day response (SD); (3) during refractoriness to short days (RSD); and (4) during inhibition by long days (ILD). LH in blood samples collected twice a week remained low during long days (0.59 +/- 0.03; mean +/- SEM), increased 45 +/- 1.5 days after the onset of short days and decreased 132 +/- 4.9 days later when ewes became refractory to short days, whereas ewes subjected to long days after 91 short days stopped their neuroendocrine activity 19 days earlier (113 +/- 4.7) (P < 0.01). In comparison with the pre-injection period, pimozide significantly increased the mean number of pulses in SD and RSD ewes, but not in LD and ILD ewes: SD: 0 versus 0.45 pulses in 4 h (P < 0.02); RSD: 0 versus 0.9 (P = 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of certain diuretics on thyroid function in rats

The effects of isolated protein fractions from rabbit uteri (prealbumin, albumin, uteroglobin, and beta-glycoprotein), unfractionated uterine proteins, progesterone, oestradiol-17beta, and prostaglandin F-2a on the development of rabbit embryos in vitro were investigated. When exposed to individual protein fractions obtained from Day-6 uteri, 8-cell embryos did not develop into early blastocysts; morulae readily developed into early blastocysts, but further development was retarded. Progesterone (10(-5)-10(-11)M) and prostaglandin F-2a (0-1-10 ng/ml) added to the medium slowed development of blastocysts to advanced stages. Growth of 8- to 16-cell embryos, morulae, and Day-4 blastocysts was stimulated by unfractionated uterine proteins obtained from Day-5 uterine flushings. Although embryos cultured in medium containing BSA had similar rates of blastocyst formation and, ultimately similar blastocyst expansion as did the embryos cultured in medium with unfractionated proteins, the radial and immediate expansion of the early blastocysts cultured in the latter approximated that found in utero.     

Hormonal profile during the follicular phase in cycles stimulated with a combination of human menopausal gonadotrophin and gonadotrophin-releasing hormone antagonist (Cetrorelix)

A third generation gonadotrophin-releasing hormone antagonist (Cetrorelix) was used during ovarian stimulation in 32 patients undergoing assisted reproduction, in order to prevent the premature luteinizing hormone (LH) surge. In all patients, ovarian stimulation was carried out with two or three ampoules of human menopausal gonadotrophin (HMG), starting on day 2 of the menstrual cycle. In addition, 0.5 mg of Cetrorelix was administered daily from day 6 of HMG treatment until the day of ovulation induction by human chorionic gonadotrophin (HCG). A significant drop in plasma LH concentration was observed within a few hours of the first administration of Cetrorelix (P < 0.005). Moreover, no LH surge was detected at any point in the treatment period in any of the 32 patients. A mean oestradiol concentration of 2111 +/- 935 ng/l was observed on the day of the HCG administration, indicating normal folliculogenesis. Like LH, progesterone concentration also dropped within a few hours of the first administration of Cetrorelix (P < 0.005). A 0.5 mg daily dose of Cetrorelix prevented a premature LH surge in all the 32 patients treated.     

Pulsatile release of LH and secretion of ovarian steroids in sheep during the luteal phase of the estrous cycle

The concentration of LH and progesterone in jugular venous plasma and the secretion of steroids by the ovary were measured every 10 minutes for 2 hours on days 12, 14, and 16 of the estrous cycle in 5 ewes with utero-ovarian autotransplants. A pulse of LH occurred about once every 2 hours, when the concentration rose from a basal level of 0.57 +/- 0.08 ng/ml to a peak of 2.97 +/- 0.57 ng/ml. Within 5 minutes of the pulse of LH, the secretion of estradiol (an exclusive product of the follicle) rose rapidly from a basal level of 0.75 +/- 0.12 ng/min to reach a peak value of 2.16 +/- 0.33 ng/min in about 30 minutes. In contrast, the secretion of progesterone from the corpus luteum, and the concentration of progesterone in the peripheral plasma changed very little following the pulse of LH. The secretion of androstenedione, which arises from the follicle and corpus luteum, increased from 3.03 +/-0.75 ng/min to 7.85 +/- 1.78 ng/min by 30 minutes after the pulse of LH. These findings indicate that the follicle, and possibly the stroma, respond rapidly to episodic fluctuations in the concentration of LH and are probably involved in the negative feedback loop between the ovary and the hypothalamic pituitary system. The fluctuations in the secretion of progesterone from the corpus luteum, on the other hand, are unrelated to pulses of LH.     

Effect of 48 h treatment with 17 beta-oestradiol or progesterone on follicular wave emergence and synchrony of ovulation in Bos indicus cows when administered at the end of a period of progesterone treatment

The objective of this study was to determine the effect of treatment with additional progesterone (P4) or 17 beta-oestradiol (E2) at the end of a period of P4 treatment on ovarian follicular development, ovulation time, and plasma gonadotrophin and steroid hormone concentrations of Bos indicus cows. Initially, two injections of PGF2 alpha were given 14 days apart, and at the time of the second injection (Day 0) all cows received a single P4-releasing controlled internal drug release (CIDR) device that was removed 10 days later. Control cows (Group 1, n = 8) received no other treatment. On Day 8, cows in Group 2 (n = 8) and Group 3 (n = 8) received either a s.c. implant containing E2, or a second CIDR device, respectively. All CIDR devices and E2 implants were removed at a similar time on Day 10. Treatment with E2 or P4 delayed mean (+/- SD) time of ovulation (113.1 +/- 25.6 h, 153.4 +/- 44.5 h and 150.8 +/- 25.1 h for Groups 1, 2 and 3, respectively; P < 0.05) and the mean time (+/- SD) of the luteinising hormone (LH) peak (87.4 +/- 24.5 h, 124.3 +/- 45.0 h and 122.3 +/- 25.04 h for Groups 1, 2 and 3, respectively; P < 0.05). Both treatments delayed the mean (+/- SD) day of emergence of the ovulatory follicle (7.7 +/- 3.6 days, 11.3 +/- 1.7 days and 11.1 +/- 1.5 days for Groups 1, 2 and 3, respectively; P < 0.05), and reduced the variability in the day of emergence of the ovulatory follicle (P < 0.05) compared with the control cows. Variability in age and duration of dominance of the ovulatory follicle was greater in control animals compared with treated animals (P < 0.05). Treatment with E2 on Days 9 and 10 did not alter mean concentrations of gonadotrophins in the cows in Group 2 compared with control cows (P > 0.05), whereas treatment of cows with an additional CIDR device resulted in greater mean concentrations of FSH and lesser concentrations of LH on Day 9 (P < 0.05) compared with cows in Groups 1 and 2. By Day 10 mean concentrations of gonadotrophins were similar among cows in all three groups. Concentrations of E2 were less in cows in Group 3 compared with cows in Groups 1 and 2 from Day 9 to Day 11 (P < 0.05). We conclude that treatment with either E2 or P4 can influence the pattern of ovarian follicular development and ovulation in cattle; however, the mechanism of action of the two treatments may differ. Atretogenic treatments for ovarian follicles applied at the end of a period of progesterone treatment did not improve synchrony of ovulation.     

Succinate and malate improve development of hamster eight-cell embryos in vitro: confirmation of viability by embryo transfer

The in vitro development of hamster preimplantation embryos is supported by non-glucose energy substrates. To investigate the importance of embryonic metabolism, influence of succinate and malate on the development of hamster 8-cell embryos to blastocysts was examined using a chemically defined protein-free modified hamster embryo culture medium-2 (HECM-2m). There was a dose-dependent influence of succinate on blastocyst development; 0.5 mM succinate was optimal (85.1% +/- 3.9 vs. 54.5% +/- 3.5). In succinate-supplemented HECM-2m, blastocyst development was reduced by omission of lactate (68.5% +/- 7.2), but not pyruvate (85.8% +/- 6.2) or glutamine (84.1% +/- 2.1). Succinate along with either glutamine or lactate or pyruvate poorly supported blastocyst development (28%-58%). Malate also stimulated blastocyst development; 0.01 mM malate was optimal (86.3% +/- 2.8). Supplementation of both succinate and malate to HECM-2m supported maximal (100%) blastocyst development, which was inhibited 4-fold by the addition of glucose/phosphate. The mean cell numbers (MCN) of blastocysts cultured in succinate-supplemented HECM-2m was higher (28.3 +/- 1.1) than it was for those cultured in the absence of glutamine or pyruvate (range 20-24). The MCN was the highest (33.4 +/- 1.6) for blastocysts cultured in succinate-malate-supplemented HECM-2m followed by those in succinate (28.3 +/- 1.1) or malate (24.7 +/- 0.5) supplemented HECM-2m. Embryo transfer experiments showed that 29.8% (+/- 4.5) of transferred blastocysts cultured in succinate-malate-supplemented HECM-2m produced live births, similar (P > 0.1) to the control transfers of freshly recovered 8-cells (33.5% +/- 2.0) or blastocysts (28.9% +/- 3.0). These data show that supplementation of succinate and malate to HECM-2m supports 100% development of hamster 8-cell embryos to high quality viable blastocysts and that non-glucose oxidizable energy substrates are the most preferred components in hamster embryo culture medium.