A correlation between ovarian follicular maturity and anchorage-independent growth of bovine granulosa cells.

Previous studies have shown that a subset of bovine ovarian granulosa cells can proliferate to form clones of functional cells in suspension in a semisolid agar matrix, without the requirement for attachment to the substratum. These clonogenic anchorage-independent granulosa cells are responsive to epidermal growth factor and exhibit properties of a primitive progenitor cell population. We have used this assay system to analyze the proliferation of granulosa cells during ovarian follicular maturation. As the follicle increases in size, there is a progressive decline in the ability of granulosa cells to clone in agar, and the proliferative potential of these cells as measured by colony size also decreases. The ratio of large colonies with high proliferative potential (greater than 250 micron in diameter) to small colonies with limited capacity for growth falls from 1.92 in follicles less than 7 mm in diameter, to 0.32 in follicles larger than 10 mm in diameter. This occurs as the follicular content of granulosa cells with more limited capacity for clonal growth in agar undergoes expansion. Analysis of colony-forming cells in follicles harvested at early and late estrus suggests that these cells are regulated by complex intraovarian factors rather than circulating gonadotropin levels. Our data indicate that the granulosa cell lineage is an age-structured continuum of maturing and differentiating cells with a progressively restricted proliferative capacity.

Role of intrafollicular regulators and FSH in growth and development of large antral follicles in sheep.

Manipulation of circulating concentrations of hormones and ovarian follicle status was carried out on Day 11-12 of the oestrous cycle in sheep. All follicles visible on the ovary were ablated by cautery and ewes were treated with oestradiol or ovine follicular fluid (oFF) to suppress FSH or with PMSG to increase circulating gonadotrophic activity. One group underwent unilateral ovariectomy which greatly increased endogenous FSH and was the only treatment which significantly affected LH pulse frequency. The size distribution of antral follicles, the extent of atresia and the mitotic index of granulosa cells of follicles on Day 15 showed that (a) treatment with oFF inhibited the growth of follicles beyond 2 mm diameter by suppressing the mitotic index of the granulosa cells and (b) the concentration of FSH in peripheral plasma was related to the ability of small antral follicles to grow during the late luteal-early follicular phase of the cycle. Subsequently, it was demonstrated that oFF inhibits, in a dose-dependent manner, folliculogenesis sustained by PMSG in ewes on Days 12-15. Inhibition of folliculogenesis was represented by a decrease in those follicles greater than 4 mm, an increase in the relative proportion of follicles less than 2 mm, and minimal change in the average number of follicles visible on the ovarian surface, and a decrease in the mitotic index of granulosa cells of follicles less than 2 mm. There was no change in the extent of atresia.(ABSTRACT TRUNCATED AT 250 WORDS)

Seasonal changes in LH secretion in normal ewes and ewes which grazed oestrogenic clover.

Plasma luteinizing hormone (LH) concentrations were measured in normal (control) Corriedale X Merino (comeback) ewes and in clover-infertile comeback ewes which had grazed oestrogenic Yarloop clover (Trifolium subterraneum L. cv. Yarloop) for more than 4 years. Plasma LH concentrations were measured in samples taken at 20-min intervals for 6 h during the dioestrous stage of the oestrous cycle in the breeding season (BS) and during the anoestrous season (AS). In the control ewes during BS, transitory elevation in plasma LH concentration (pulses) occurred, reflecting secretory episodes, with a frequency of one per 5.2 h. This frequency fell to one per 16.5 h during the anoestrous season. In clover-infertile ewes, LH pulses occurred with a frequency of one per 4.5 h during BS and one per 4.9 h during AS (difference not significant). In the controls, plasma LH levels were higher (P less than 0.05) during BS (mean +/- s.d. = 1.2 +/- 0.4 ng/ml, n = 9) than in AS (0.7 +/- 0.3 ng/ml, n = 5). In the clover-infertile ewes, plasma LH levels in BS (1.3 +/- 0.6 ng/ml, n = 12) were similar to those of controls. During AS, plasma LH levels in the clover-infertile ewes (1.0 +/- 0.6 ng/ml, n = 10) remained similar to their BS levels, being significantly (P less than 0.05) higher than LH levels in the controls at this time. These studies indicate that the higher plasma concentrations of LH which have been reported in clover-infertile ewes arise from more frequent LH pulses. Furthermore, in contrast to normal ewes, average plasma LH, reflecting pulse frequency, is not reduced in AS. This supports the view that ingestion of phytooestrogens affects neural centres involved in regulating LH secretion.

Relaxin inhibits the plateau component of the action potential in the circular myometrium of the rat.

The effects of relaxin on contractility and membrane potential of the longitudinal and circular smooth muscle layers of the uterus have been studied in vitro using oestrogen-treated, non-pregnant rats and pregnant rats. Relaxin decreased the amplitude of contractions induced by electrical stimulation of longitudinal myometrium by decreasing the duration of the bursts of action potentials. This effect was transient and tachyphylaxis always developed and was observed following injection of steroids and up to day 17 of pregnancy. There was no inhibition of tissues from rats from day 18 of pregnancy to term. The peptide had no effect on resting membrane potential, space constant or time constant. Action potentials recorded from circular myometrium of non-pregnant rats pre-treated with oestrogen consisted of an initial spike or short burst of spikes followed by a prolonged plateau of depolarization. Spontaneous action potentials and associated contractions were abolished within 2 min of exposure to relaxin (10(-8) g/ml) while contractions of much smaller amplitude could be evoked with depolarizing current pulses. This effect was associated with depression of the plateau component of the action potential whereas the spike component was left intact. Relaxin had no effect on passive membrane properties. The action potentials of circular myometrium of rats up to day 21 of pregnancy were qualitatively similar to those recorded in the same muscle layer from oestrogen-treated, non-pregnant rats and the plateau component was also blocked by relaxin in these tissues. Bursts of spikes were observed in circular strips 24-36 h before parturition, and the effect of the peptide on these was a transient inhibition similar to that observed in longitudinal myometrium. Oxytocin increased the amplitude of the spike and the amplitude and duration of the plateau. Relaxin abolished the plateau in the presence of 10(-11) and 10(-10) M-oxytocin but was ineffective when the concentration of the spasmogen was increased further. Prostaglandin F2 alpha increased the amplitude and duration of the plateau. Relaxin abolished the responses to 10(-10) and 10(-9) M-prostaglandin F2 alpha. The results of this study demonstrate that relaxin specifically inhibits contractions in the circular layer of the myometrium by abolishing the plateau component of the action potential. This action appears to be different from that of other smooth muscle relaxants tested in these experiments (isoprenaline, papaverine and verapamil). All of these abolished simultaneously both the spike and plateau components of the action potential.

Differences in the reproductive endocrine status of ewes in the early post-partum period and during seasonal anoestrus.

More (P less than 0.05) post-partum acyclic ewes (8/9) showed evidence of pulsatile LH release than did seasonally anoestrous ewes (2/8). Mean plasma prolactin concentrations were higher (P less than 0.05) in the post-partum ewes than in the seasonally anoestrous ewes. Hypothalamic and pituitary cytosol oestrogen receptor number, median eminence GnRH content and pituitary LH, FSH and prolactin contents were similar in the two groups of ewes. The number of nuclear-bound oestrogen receptors was greater (P less than 0.01) in pituitaries from seasonally anoestrous ewes than in pituitaries from post-partum ewes. These data suggest that the basis of acyclicity in seasonally anoestrous ewes and in post-partum ewes is probably due to their inability to generate LH pulse frequencies similar to that in the follicular phase of the oestrous cycle. The higher LH pulse frequency in post-partum ewes, compared to that in seasonally anoestrous ewes, suggests that pregnancy may partly negate the reduction in LH secretion that is associated with photoperiod-induced acyclicity. The lower number of nuclear-bound oestrogen receptors in post-partum ewes suggests that there may be lower plasma oestrogen levels and less ovarian follicular growth than in non-suckling anoestrous ewes.

The characteristics of an anchorage-independent clonal agar assay for primary explanted bovine granulosa cells.

Normal, primary explanted, bovine granulosa cells grow reproducibly in agar culture as anchorage-independent clones. Epidermal growth factor (EGF) and rat erythrocytes are effective stimulators of colony formation, and when both are added to the culture medium at optimal concentrations, there is an enhancement of colony numbers and colony size, indicative of an independent, and operationally additive, mode of action for the two factors. The ability of cells propagated from agar clones to secrete progesterone, and to augment progesterone secretion 4-fold in the presence of 1 mM dbcAMP is proof that colonies originate from and are composed of functional granulosa cells. Maximal colony numbers are present at day 10 of incubation, and colony forming cells undergo self-renewal as assessed by the ability of cells from primary colonies to reclone in agar. Absolute cloning efficiency, however, is dependent on a number of factors. Inherent variability exists in cloning efficiency of granulosa cells from individual follicles. Quantitative and qualitative clonal growth was improved at an osmolality of less than 300 mOsm when compared with higher osmolalities. Cl-1 medium and the alpha modification of Eagle's medium were equally effective in supporting agar clonogenic growth, whereas both Ham's F12 and NCTC 135 media exhibited poor clonogenic growth supporting properties. The substitution of agarose for agar did not affect colony numbers but colonies grown in the presence of agarose tended to be smaller and more uniform in size.

The time of oestrus and ovulation following various synchronisation techniques using progesterone impregnated intravaginal devices.

Progesterone releasing intravaginal devices (PRIDs) were used in 4 experiments involving 67 cattle to study the effect of the stage of the oestrous cycle and of oestradiol benzoate (ODB) administration on the time interval from PRID removal to oestrus and/or ovulation. Cows in which PRIDs were inserted on days 2 to 4 of the oestrous cycle for 14 days were subsequently observed by endoscopy to ovulate significantly later than cows given identical treatments on days 13 and 14 of the cycle. The concentration of progesterone was higher in the former group at the time PRIDs were removed and remained at a higher level for 3 days thereafter. The length of the oestrous cycle of untreated heifers was significantly longer than in heifers treated for 12 days with PRIDs only inserted on day 3 of cycle (19.7 v 18.2; p less than 0.01). Treatment with PRIDs inserted for 12 days together with ODB further shortened the cycle length from 18.2 days to 16.9 days (p less than 0.01). The intervals from PRID removal to onset of standing oestrus in heifers treated with PRID only and those treated with PRID and ODB were 80h and 45h, respectively (p less than 0.01); the respective variances were 280 and 4; p less than 0.01). Plasma progesterone concentration, at PRID removal, was 3 times higher in heifers treated with PRID only than in heifers treated with PRID and ODB (p less than 0.01) and the interval from PRID removal to the lowest progesterone value following it was 3.8 ad 1.2 days for the 2 groups, respectively (p less than 0.05). The study indicated that the stage of oestrous cycle at PRID insertion is an important factor which influences the interval from withdrawal of PRIDs to ovulation. The results suggest closer synchronisation would follow insertion in the mid-luteal phase (Day 13 to 14) or use of ODB treatment at the time of insertion of PRIDs.

Hypothalamic-pituitary function in normal ewes and ewes which grazed oestrogenic subterranean clover for several years.

Plasma luteinizing hormone (LH) concentrations were measure in normal Merino ewes and in Merino ewes with lowered fertility which had resulted from prolonged grazing of Trifolium subterraneum L. cv. Dinninup. During the anoestrous season, LH was measured at frequent intervals before and following administration of oestradiol-17 beta or gonadotropin releasing hormone (GnRH), and around the time of spontaneous oestrus. All ewes responded to GnRH treatment and there were no differences between the two groups in terms of the amount of LH released or the time to reach maximal plasma hormone concentration. Comparable proportions of ewes from both groups responded to oestradiol-17 beta treatment with LH levels showing initially a negative feedback response (i.e. suppression to approximately 80% of pretreatment levels) followed by positive feedback response. During the positive feedback phase, the quantity of LH which was released was comparable in both groups; however, the time interval over which this hormone was secreted was significantly shorter for clover-infertile ewes (P less than 0.05). During the anoestrous season the mean LH level was lower in normal ewes than in clover-infertile ewes [0.45 +/- 0.41 (n = 32) versus 0.82 +/- 0.73 (n = 48 ng/ml, P less than 0.01]. In the oestrous season the mean LH level for normal ewes rose to 1.23 +/- 0.65 ng/ml (n = 76) but there was no change for the clover-infertile ewes (0.77 +/- 0.59 ng/ml, n = 73). These experiments indicate that in the clover-infertile ewe LH concentration remains static throughout the breeding and non-breeding seasons but the results suggest that this altered endocrine status in not due to any differing level of sensitivity to negative feedback effects of oestradiol-17 beta as compared with normal ewes.

Potencies of porcine relaxins using two bioassays.

Relaxin reference preparations NIH-R-P1 and Warner Lambert W1164-3, and purified relaxin peptides, CM-a', CM-a and CM-B, were assayed in the mouse interpubic ligament and rat uterine inhibition bioassays. There was evidence that CM-a and CM-B would bind to glass and significant apparent increases in potency in the case of these two peptides alone resulted from the use of silicone-coated glassware. Using treated glassware, CM-a', CM-a and CM-B were equipotent in the mouse interpubic ligament bioassay with potencies relative to NIH-R-P1 of 3.97-, 4.85- and 3.64-fold respectively. In the rat uterine inhibition bioassay only W1164-3 and CM-a' gave dose-response curves which were parallel to NIH-R-P1. In this bioassay, potencies relative to NIH-R-P1 for W1164-3 and CM-a' were 0.35- and 19.6-fold respectively.

Critical time period for the vagino-cervical stimulus to induce pseudopregnancy in the dioestrous rat.

Groups of rats experienced mechanical stimulation of the vagino-cervical region in induce pseudopregnancy at various intervals from 0900 h on dioestrus -- 1 (D -- 1) to 1800 h on D -- 2. Twenty-four of 26 rats which were stimulated on D -- 1 experienced immediate pseudopregnancy whereas when the stimulus was given on D -- 2, 39 of 45 rats continued to cycle and the latest time when such mechanical stimulation would reliably induce immediate pseudopregnancy was 2400 h on D -- 1. Abolition of the dark phase between 1800 h D -- 1 and 0600 h D -- 2 did no alter this critical time period (2400 h on D -- 1). Stimulation as late as 1200 h on D -- 2 caused alterations in the normal pattern of 4-day cyclicity in 8 of 11 rats.

Pituitary responsiveness to LH-RH in post-partum ewes treated with oestradiol-17 beta and failing to show a plasma LH surge.

Oestradiol-17 beta (40 micrograms intravenously) failed to elicit a surge in plasma LH levels by 13 h after administration in 64% (16 out of 25) Merino ewes about 30 days post partum in the anoestrous season. LH-RH responsiveness and LH-RH priming effect were significantly greater in these ewes than in similar post-partum (n = 9) and non-parturient ewes (n = 3) not treated with oestradiol. This suggests that the failure of the oestrogen-positive feedback effect on LH release in post-partum ewes is not due to a failure of oestradiol action on the pituitary increasing pituitary responsiveness to LH-RH and LH-RH priming effect, but could be due to inadequate release of LH-RH from the hypothalamus.

Identification of relaxins in porcine follicular fluid and in the ovary of the immature sow.

Relaxin was measured by radioimmunoassay in follicular fluid from sows which were of different reproductive states. In non-pregnant animals, follicular fluid relaxin values ranged from 0.23 to 666 ng/ml. For pregnant animals the range was 172.6-32 707 ng/ml and for sows with polycystic ovaries this range was 5.0-2800 ng/ml. For polycystic ovaries only there was a correlation (r = 0.74) between the diameter of the follicle and relaxin concentration in the respective fluid. Gel filtration species of relaxin, of molecular weights 27500, 19000 and 6000. Relaxin was extracted from 1 kg of ovaries collected from prepubertal pigs and gel filtration studies indicated the presence of large molecular weight species in addition to a molecule of approximately 6000. These experiments suggest that the corpus luteum is not the sole source of relaxin in the pig.

Plasma relaxin immunoactivity in the pig at parturition and during nuzzling and suckling.

One sow bled at 30--60-min intervals for 48 h at 5 and 4 days before parturition had mean +/- s.e.m. relaxin levels of 5.0 +/- 0.48 ng/ml and 5.5 +/- 0.44 ng/ml for each 24-h period respectively. This sow and another were bled at frequent intervals during parturition; both showed considerable fluctuations in their relaxin levels but no consistent peaks in relation to each birth. Mean levels during parturition were 10.7 +/- 0.46 ng/ml and 13.4 +/- 0.81 ng/ml respectively, both significantly higher than the levels at 4 and 5 days before birth. Relaxin levels in two lactating sows rose acutely during nursing, showing a 3-fold rise in one animal and an 8-fold rise in the other. Results from a third sow during an extended period of nuzzling and sucking by the piglets showed multiple peaks of relaxin immunoactivity associated with each nuzzling/sucking stimulus.

Pituitary response to LHRH during the puerperium in women.

Five breast-feeding women received 100 mug synthetic LHRH at between 19 and 58 days post-partum. One subject was treated twice. LH and FSH responses were monitored using radioimmunoassay. In four women treated at 19,21,29 and 51 days post-partum, the FSH response was greater than the LH response which was absent altogether in three of these women. In two women who were treated at 52 and 58 days both LH and FSH levels was elevated with the LH response being greater than the FSH response. This change in the pattern of pituitary response is considered in relation to the post-partum anovulatory condition.

Gonadotrophin levels and ovarian development in the neonatal ewe lamb.

Ovarian weight has been shown to increase markedly in the newborn ewe lamb, and factors which might contribute to this growth were examined. Follicle development was studied in the ovaries of 28 lambs aged 0, 2, 4, 6, 8 and 10 weeks. Plasma samples, pituitaries and one ovary were assayed for gonadotrophin or steroid hormone content. No significant differences between age groups were found in pituitary LH concentration or mean plasma LH. Pituitary FSH concentration increased with age, except for a small decrease at 8 weeks, but no significant changes were seen in plasma FSH. Ovarian progesterone and oestradiol concentrations did not appear to be associated with follicular development. Growing follicles were most numerous at 2 weeks whilst total vesicular follicles reached a peak at 4 weeks. Most vesicular follicles were less than 1.4 mm in diameter. Advanced atresia in vesicular follicles became apparent by 4 weeks although early signs were present in younger lambs. Fluctuations in gonadotrophin levels do not appear to be responsible for variations in number and size of vesicular and growing follicles in the lamb ovary. Other possible explanations are discussed.

Effect of prolactin on the LH response to synthetic LH-RH in ovariectomized ewes.

When ovariectomized ewes were treated with LH-RH, all 3 receiving prolactin infusion and 4 out of 5 receiving an infusion of NaCl solution responded.

Prolactin response to synthetic TRF in pregnant ewes.

The prolactin secretion response to TRF was similar at all stages of pregnancy (Days 40, 66, 102 and term).