Reproduction

Dobson, H.; Tebble, J. E.; Phogat, J. B.; Smith, R. F.

doi: 10.1530/jrf.0.1160001pmid: 10505050

The aim of this study was to elucidate the mechanism(s) involved in stress-induced subfertility by examining the effect of 4 h transport on surge and pulsatile LH secretion in intact ewes and ovariectomized ewes treated with steroids to induce an artificial follicular phase (model ewes). Transport caused a greater delay in the onset of the LH surge in nine intact ewes than it did in ten ovariectomized ewes (intact: 41.0 ± 0.9 h versus 48.3 ± 0.8 h, P < 0.02; ovariectomized model: 40.8 ± 0.6 h versus 42.6 ± 0.5 h, P < 0.02). Disruption of the hypothalamus–pituitary endocrine balance in intact ewes may have reduced gonadotrophin stimulation of follicular oestradiol production which had an additional effect on the LH surge mechanism. In the ovariectomized model ewes, this effect was masked by the exogenous supply of oestradiol. However, in these model ewes, there was a greater suppression of maximum LH surge concentrations (intact controls: 29 ± 4 ng ml−1 versus intact transported 22 ± 5 ng ml−1, P < 0.02; ovariectomized model controls: 35 ± 7 ng ml−1 versus model transported 15 ± 2 ng ml−1, P < 0.02). Subsequent exposure to progesterone for 12 days resulted in the resumption of a normal LH profile in the next follicular phase, indicating that acute stress leads to a temporary endocrine lesion. In four intact ewes transported in the mid-follicular phase, there was a suppression of LH pulse amplitude (0.9 ± 0.3 versus 0.3 ± 0.02 ng ml−1, P < 0.05) but a statistically significant effect on pulse frequency was not observed (2.0 ± 0.4 versus 1.7 ±0.6 pulses per 2 h). In conclusion, activation of the hypothalamus–pituitary–adrenal axis by transport in the follicular phase of intact ewes interrupts surge secretion of LH, possibly by interference with LH pulsatility and, hence, follicular oestradiol production. This disruption of gonadotrophin secretion will have a major impact on fertility.

Phogat, J. B.; Smith, R. F.; Dobson, H.

doi: 10.1530/jrf.0.1160009pmid: 10505051

This study examined the effect of transport on GnRH self-priming in vivo as well as the consequential effects on the oestradiol-induced LH surge. The follicular phases of ewes (eight per group) were synchronized with progestin sponges, and 50 μg oestradiol benzoate was injected 24 h (time zero) after sponge removal to improve precision in the timing of the LH surge. Beginning 8 h after oestradiol, saline or GnRH (500 ng, i.v.) was given at 2 h intervals with or without 8 h transport beginning 0.5 h before the first GnRH injection (late transport) and effects were compared with those observed during early transport, that is, starting 2.5 h before the first GnRH injection. In all ewes, GnRH alone induced a maximum LH response of 1.9 ± 0.4 ng ml−1 after the first challenge. The response was enhanced (P < 0.01) after the second and third GnRH injections (7.4 ± 1.4 ng ml−1 and 7.6 ± 1.7 ng ml−1, respectively). This self-priming effect after the second GnRH was reduced by late transport (7.4 ± 1.4 versus 4.2 ± 0.8 ng ml−1; P < 0.05) but not early transport, that is, transport initiated closer to the time of GnRH administration had greater suppressive effects on LH secretion. Throughout transport, spontaneous LH pulse frequency was lower in treated than it was in control ewes (2.38 ± 0.53 versus 4.50 ± 0.53 pulses per 8 h; P < 0.01), with marked effects in the first 4 h of transport (1.0 ± 0.19 versus 2.63 ± 0.38 pulses per 4 h; P < 0.02). Spontaneous pulse amplitude also tended to decrease during transport (0.13 ± 0.02 versus 0.20 ± 0.03 ng ml−1; P = 0.07). When LH turnover was stimulated by exogenous GnRH, the onset of the LH surge was delayed (controls: 20.5 ± 2.0 h versus GnRH alone: 25.3 ± 1.5 h; P < 0.05) and the duration was reduced (8.5 ± 0.9 versus 6.5 ± 0.4 h; P < 0.05). Transport tended to delay the LH surge in saline-treated ewes (20.5 ± 2.0 versus 22.9 ± 1.9 h; P = 0.08), with a further delay imposed by late transport plus GnRH (27.5 ± 1.6 h; P < 0.05) but not by early transport plus GnRH (27.8 ± 2.5 versus 26.4 ± 2.4 h; P > 0.05), that is, effects mediated by increasing LH turnover were only manifest if transport occurred near the LH surge, when there was insufficient time to replenish stores of releasable LH. In all transported ewes, plasma cortisol increased from 4.5 ± 1.0 ng ml−1 to 29.2 ± 5.5 ng ml−1 (P < 0.001) within 15 min of the start of transport and was significantly lower (P < 0.01) by 6.5 h. Plasma progesterone also increased from 0.30 ± 0.04 to 0.38 ± 0.04 ng ml−1 (P < 0.05). In conclusion, transport affected the oestradiol-induced LH surge by causing a 50% reduction in the self-priming effect of exogenous GnRH, but hypothalamic effects were also revealed by a two-fold decrease in spontaneous LH pulse frequency in saline-treated ewes.

Jiang, J. Y.; Miyoshi, K.; Umezu, M.; Sato, E.

doi: 10.1530/jrf.0.1160019pmid: 10505052

The aim of this study was to examine the effect of thyroxine on ovulation in immature rdw rats and the fertilization and development of the eggs. Serum thyroxine concentrations at 30 days of age were significantly lower in rdw rats than in normal rats (P < 0.001), and greatly increased after thyroxine replacement therapy (P < 0.001). Although few eggs (1–5 ± 1–2) were obtained from immature rdw rats treated with gonadotrophins alone, females treated with gonadotrophins and thyroxine ovulated significantly more eggs (85 ± 5). As a control, normal littermates ovulated 21–45 eggs when treated with gonadotrophins alone, and 68 eggs when administered with gonadotrophins and thyroxine. Of the eggs collected from rdw rats treated with gonadotrophins and thyroxine, and inseminated with spermatozoa from mature F1 males, 98% were penetrated and in almost all (99%) of these eggs, male and female pronuclei formed. Forty-seven per cent of the pronuclear eggs developed to the blastocyst stage in vitro. After transfer to recipients, 21% (14/66) of one-cell and 22% (8/37) of two-cell embryos developed to offspring, and 62% (8/13) of pups were of rdw/rdw genotype. The average body weight (6.9 versus 7.8 g) of offspring derived from one-cell embryos was lower than that for two-cell embryos. The morulae and blastocysts did not develop to term, although 41% implanted in the uterine horns of recipients. In conclusion, in immature rdw rats, superovulation was induced by gonadotrophins combined with thyroxine therapy and the superovulated oocytes were fertilized and developed in vitro and developed to term after embryo transfer.

Gebbie, F. E.; Forsyth, I. A.; Arendt, J.

doi: 10.1530/jrf.0.1160025pmid: 10505053

Interactive effects of light and temperature on aspects of seasonality were studied in female British Saanen dairy goats. Four groups of adult non-pregnant non-lactating goats (n = 5) were housed under the following conditions: controls (July–June): natural photoperiod and temperature; group 1 (July–December): long days (16 h light: 8 h dark) and natural temperature; group 2 (July–December): long days and average summer temperature (17.6°C); group 3 (December–June): short days (8 h light: 16 h dark) and winter temperature (8.4°C). Plasma prolactin and progesterone were measured once a week, circadian changes in prolactin and melatonin were determined in December and May, and coat development was assessed. Seasonal variation in prolactin was influenced by manipulation of both daylength and temperature. In group 1, prolactin concentrations decreased as the environmental temperature decreased, despite maintenance of long days. When light and temperature were maintained under summer (group 2) and winter (group 3) conditions, prolactin remained relatively constant, although at different high and low set points, respectively, but with indications of a seasonal rhythm. An asymptotic relationship between prolactin and temperature was maintained under all daylengths. The circadian pattern of melatonin was related to daylength and was not influenced significantly by temperature. Onset of oestrus was unaltered. In group 3 (maintained winter solstice light and temperature), anoestrus was delayed (P < 0.05) from a median control date of 17 March to a median date of 28 April. Winter coat development was delayed in group 1; group 2 showed premature moulting of the winter coat; and in group 3, development of the summer coat was delayed. The results imply that temperature modifies the influence of daylength on prolactin secretion and hair follicle growth by mechanisms that do not involve melatonin.

Hynes, A. C.; Kane, M. T.; Sreenan, J. M.

doi: 10.1530/jrf.0.1160035pmid: 10505054

Granulosa cell-inhibitory factor (GCIF), a low molecular weight factor from bovine follicular fluid, inhibits the proliferation of bovine granulosa cells in vitro and the growth of large follicles in rats in vivo. In this study the effects of (1) immunization of rats against GCIF on follicular growth and (2) immunization of sheep against GCIF on ovulation rate were studied. The ability of antiserum from sheep immunized against GCIF to reduce the inhibitory effect of GCIF on bovine granulosa cell proliferation in culture was also examined. Immunization of rats against GCIF increased the number of large follicles (P < 0.001) but decreased the number of small follicles (P < 0.05) per ovary. Ovarian mass (P < 0.05) and uterine wet (P < 0.05) and dry (P < 0.01) masses were increased in immunized rats. Immunization of sheep against GCIF, followed by boosting over two breeding seasons, increased ovulation rate (P < 0.01). Addition of antiserum from sheep immunized against GCIF reduced or abolished the inhibitory effect of GCIF on granulosa cell proliferation (P < 0.01). These data provide further evidence that GCIF has an important role in controlling follicle growth and ovulation in vivo.

Tsatas, D.; Baker, M. S.; Rice, G. E.

doi: 10.1530/jrf.0.1160043pmid: 10505055

A number of tightly regulated proteolytic enzyme systems, including the plasminogen activation cascade and matrix metalloproteases, play integral roles in the remodelling of extracellular matrices during pregnancy and parturition. This study assessed these labour-associated changes in protease activity in human gestational tissues. Amnion, choriodecidua and placenta collected from women before (at caesarean section, not in labour), during (at caesarean section, in labour) and after (spontaneous-onset labour, normal vaginal delivery) labour were examined on gelatin-substrate SDS-PAGE zymography. All tissues displayed major 55 kDa plasminogen-dependent activity that was abolished by the serine protease inhibitors (10 mmol phenylmethyl-sulphonylfluoride l−1, 100 mmol epsilon aminocaproic acid l−1, 1 mmol Glu-Gly-Arg chloromethylketone l−1). The enzymic activity was identified as urokinase plasminogen activator on the basis of its co-migration with reference standard and western blot analysis, and did not vary with labour status. An additional protease with an apparent molecular mass of approximately 90 kDa was detected in all tissues. Densitometric measurement of these tissues showed a significant (P < 0.05) increase in this enzyme activity with labour onset. Heavy metal chelators (1 mmol 1, 10 phenanthroline l−1 and 10 mmol EDTA l−1) selectively blocked the 90 kDa activity, consistent with the proposal that it is a metalloprotease. Co-migration with reference standard and western blot analysis confirmed the identity of this protease as the matrix metalloprotease 9 (MMP-9). Immunoreactive MMP-9 protein was also significantly (P < 0.05) increased during and after labour compared with before labour in all tissues examined. It is proposed that the upregulated expression of MMP-9 is involved in fetal membrane rupture and placental separation during and after labour onset, respectively. In conclusion, the regulated repertoire of protease activities expressed by human gestational tissues implies an important role for matrix-degrading enzymes during human parturition.

Reynaud, K.; Hanrahan, J. P.; Donovan, A.; Driancourt, M. A.

doi: 10.1530/jrf.0.1160051pmid: 10505056

High prolificacy due to a gene that has a large effect on ovulation rate has been noted in Booroola and Inverdale ewes. High prolificacy in the Belclare breed (a composite developed from stocks selected for very large litter size or high ovulation rate) may be related to the segregation of two genes. The aims of this study were (i) to compare the morphological and functional features of ovulatory follicles from carriers (which could only be heterozygous for the genes of interest) and non-carriers, and (ii) to identify markers of the Belclare genes among secreted or cellular ovarian proteins. Belclare carrier ewes had more ovulatory follicles (4.9 ± 0.4) than did non-carrier ewes (2.0 ± 0.2) (P < 0.001). Ovulatory follicles from carriers were also smaller (4.4 ± 0.1 mm versus 5.7 ± 0.2 mm, P < 0.001) and contained a significantly reduced number of granulosa cells (P < 0.001). However, the proportion of proliferating granulosa cells in ovulatory follicles was similar in both groups. The in vitro secretion of steroids per follicle was only marginally lower in follicles from Belclare carriers compared with non-carriers. Furthermore, similar concentrations of steroidogenic enzymes were present in both groups, indicating that steroidogenic potential per granulosa cell is similar between carriers and non-carriers. Possible markers of the Belclare genes were identified among cellular proteins of follicular walls by two-dimensional PAGE and image analysis. Two spots at 78 and 49 kDa were always absent in samples from non-carriers. When secreted proteins in follicles from carriers were compared with those from non-carriers, two spots at 53 and 41 kDa were restricted to samples from carriers and three spots at 97, 91 and 45 kDa were unique to samples from non-carriers. Interestingly, the spot at 91 kDa is also affected by the Booroola gene.

Tiemann, U.; Neels, P.; Pöhland, R.; Walzel, H.; Löhrke, B.

doi: 10.1530/jrf.0.1160063pmid: 10505057

Oviductal endosalpingeal cells were isolated mechanically from heifers and cultured until there was 100% confluency. The cells were loaded with the Ca2+-sensitive fluorochrome, fura-2/acetoxymethylester, and cytosolic free calcium ([Ca2+]i) was monitored by spectrofluorimetry. Platelet-activating factor, at a concentration of 30 nmol l−1, induced an intracellular Ca2+ increase in cultured bovine oviductal cells, mainly via influx from the extracellular space. In fura-2-loaded oviductal cells, different Ca2+channel blockers were investigated to characterize the pathways responsible for the Ca2+ influx. The negative effects of Ni2+-, La3+- and Ca2+-activated K+ channel blockers, such as apamin and charybdotoxin, and Ca2+ channel blockers, such as dotarizine, on the platelet-activating factor-induced [Ca2+]i increase indicate the minor participation of the voltage-gated Ca2+ channels. TMB-8 and flufenamic acid blocked the platelet-activating factor-induced Ca2+ increase directly on non-selective cationic channels or acted via a Ca2+ release-triggered Ca2+ influx. Platelet-activating factor, at concentrations of 1.25 μmol l−1 and 2.5 μmol l−1, significantly stimulated the proliferation and depolarization of oviductal cells, but 10 μmol l−1 significantly decreased both parameters and exerted a cytotoxic effect on cells. After incubation with TMB-8 or flufenamic acid, the cell proliferation was inhibited in a concentration-dependent manner, with IC50 values of 26.57 μmol l−1 and 95.29 μmol l−1, respectively. The depolarization was significantly inhibited at 50 μmol l−1 for both TMB-8 and flufenamic acid. The results of the present study may contribute to further understanding of the mechanism behind the actions of platelet-activating factor on oviductal cells.

Onagbesan, O. M.; Decuypere, E.; Leenstra, F.; Ehlhardt, D. A.

doi: 10.1530/jrf.0.1160073pmid: 10505058

Strain differences in reproductive performance were demonstrated between broiler breeder female chickens selected for growth (GL line) or for food conversion efficiency (FC line) and the improvement in reproductive performance due to feed restriction also differed significantly. Feed allowance effects on the maturation of ovarian follicles, the incidence of atresia and egg production differed between the two lines exposed to similar feeding protocols. Feed restriction reduced body weights significantly and to a similar extent in both GL and FC lines. The number of normal and atretic yellow follicles was significantly higher under ad libitum feeding and in GL line than it was in the FC line. In both lines, feed restriction decreased multiple ovulation and increased egg production. In culture, granulosa cells from the three largest follicles (F1, F2 and F3) increased progesterone production in response to LH, FSH and insulin-like growth factor I but responses were different between the GL and FC lines fed either ad libitum or restricted diets. Granulosa cells from the two or three largest follicles in GL and FC (ad libitum) lines produced similar amounts of progesterone in response to LH, FSH and insulin-like growth factor I whereas, in restricted birds, the progesterone production was of the rank order Fl > F2 > F3 in both lines. The responsiveness of the GL line fed ad libitum was higher for LH than for either FSH or insulin-like growth factor I but in the GL line fed a restricted diet, it was high for all the hormones. In the FC line, responses to LH, FSH or insulin-like growth factor I were high in ad libitum-fed birds, but low in birds fed a restricted diet for all hormones. Insulin-like growth factor I combined with LH or FSH significantly increased the progesterone production of granulosa cells from birds fed restricted diets of both lines and this effect increased with increasing follicular size. There was a lack of interaction between insulin-like growth factor I and LH or FSH in the regulation of progesterone production by birds of both lines fed ad libitum. Insulin-like growth factor alone or in combination with LH or FSH increased granulosa cell proliferation in birds fed ad libitum more than it did in birds fed restricted diets. The greater proliferation rate of granulosa cells of chickens fed ad libitum, in response to insulin-like growth factor I alone or in combination with gonadotrophins, leading to the simultaneous differentiation of two or three large follicles with high progesterone production in response to LH or insulin-like growth factor I, accelerates the rate of maturation of follicles. This may also be the major cause of erratic and multiple ovulations in broiler breeder female chickens fed ad libitum. In conclusion, insulin-like growth factor I, alone or in combination with LH or FSH, is an important component in the control mechanisms for follicular development in broiler breeder hens. It is this component that is targeted by feed allowance and inadvertently altered by selection for growth.

Holekamp, K. E.; Szykman, M.; Boydston, E. E.; Smale, L.

doi: 10.1530/jrf.0.1160087pmid: 10505059

Reproductive seasonality was examined in an equatorial population of free-living spotted hyaenas (Crocuta crocuta) in Kenya. The study population was observed continuously for 10 years, during which time the dates of all births, conceptions, weanings, and cub deaths were recorded. Local prey abundance was estimated two to four times per month, and rainfall was recorded daily throughout the study period. Births occurred during every month of the year, but a distinct trough in births occurred from February to May. This trough occurred approximately one gestation period after the phase of the annual cycle during which prey animals were least abundant in the home range of the hyaenas, and conceptions occurred most frequently when food abundance was greatest. Neither rainfall nor cub mortality were correlated with births or conceptions. Thus, although spotted hyaenas are capable of breeding throughout the year, they exhibit a moderate degree of seasonality that most likely reflects responses to seasonal variation in energy availability.