Abstract

Changes in LH secretory granules in pituitary gonadotrophs throughout the sheep oestrous cycle were determined by immunogold localisation of LH at ultrastructural level by electron microscopy. Oestrous cycles in Welsh Mountain ewes were initially synchronised with progestagen sponges and studies carried out in the subsequent cycle. Animals were allocated at random to six groups each of five animals, one killed on day 12 of the luteal phase and the other groups after prostaglandin (PG)-induced luteal regression at PG plus 18 h (early follicular phase), oestrus (PG plus 33·6±1·0 h), oestrus plus 9 h just before the preovulatory LH surge, 1 h after GnRH agonist-induced LH surge at PG plus 48 h (mid-LH surge) and oestrus plus 24 h, after the preovulatory LH surge. Blood samples collected throughout confirmed the pulsatile secretion of LH before and the timing in relation to the preovulatory LH surge. Pituitaries were dissected and processed for transmission electron microscopy and frozen for later extraction of mRNA. Only a single type of LH cell was present in the sheep pituitary. In the luteal phase, LHimmunopositive secretory granules were distributed throughout the cytoplasm in 80% of cells while in 20% of cells granules were polarised to the region of the cell next to a vascular sinusoid. The percentage of polarised cells increased during the follicular phase to 45% at oestrus, 75% at oestrus plus 9 h just before the LH surge and 90% in mid-LH surge. Cell size increased in parallel with polarisation. Gonadotrophs after the LH surge were almost totally devoid of LH granules but prominent LHβ immunoreactivity was observed in the rough endoplasmic reticulum. Analysis of granule diameters revealed a single class of granules with a maximum diameter of 300 nm. Polarised cells had significantly fewer 130–150 nm granules than non-polarised cells, suggesting preferential exocytosis of LH-containing granules of this size from polarised cells. Northern analysis showed that LHβ mRNA levels decreased from luteal through the follicular phase. These results suggest that the preovulatory LH surge in sheep is not related to a change in synthesis of LH but to a progressive recruitment of gonadotrophs into a releasing state, priming, as indicated by polarisation of secretory granules to the region of the cell next to the vascular system.

Journal of Endocrinology (1995) 147, 259–270

Abstract

We have previously demonstrated that 1·9 kb of ovine LHβ promoter fused to bacterial chloramphenicol transferase (CAT) coding sequence is sufficient to target expression of the transgene specifically to the gonadotroph cells of the anterior pituitary in mice with no expression being observed in other tissues. However, it is not known if this region of the ovine LHβ promoter contains the necessary elements that confer transcriptional regulation by gonadal steroids and GnRH. Following gonadectomy, both endogenous pituitary LH and CAT activity significantly (P>0·001) increased as did plasma LH. This postgonadectomy increase in CAT, pituitary and plasma LH could be suppressed in females by treatment with oestradiol alone or oestradiol and progesterone, with an additional significant (P<0·05) reduction in CAT activity being observed in one line following the combined steroid treatment. In castrated males, testosterone suppressed CAT activity in one line. Treatment of transgenic ovariectomized females with oestradiol alone significantly suppressed plasma LH (P<0·01) with no change in pituitary LH content. There was no difference in pituitary LH between oestradiol-treated ovariectomized transgenic and non-transgenic females. Treatment of intact females from both lines with either GnRH antiserum or agonist demonstrated a decrease in pituitary CAT activity whereas similar treatment in intact males had no effect. While endogenous pituitary LH concentrations were variable, plasma LH was lower in all treated animals irrespective of line, sex or expression of the transgene. In conclusion, these results indicate that (1) the presence of CAT protein is not toxic and does not compromise either endogenous LH synthesis, storage and secretion and (2) the ovine LHβ–CAT gene is regulated in a similar but more variable manner to the endogenous LHβ gene. This may relate to the use of CAT as a reporter where its release is not necessarily related to that of the endogenous hormone whose synthesis, storage and release may differ.

Journal of Endocrinology (1996) 151, 481–489

The plasma concentrations of FSH, LH, prolactin and progesterone were measured throughout anoestrus and during the resumption of cyclic activity in two groups of ewes. Group A was maintained under conditions of natural daylength throughout the experiment, whereas Group B was exposed to 6 h of light and 18 h of darkness, the change being made abruptly on the longest day. In those ewes kept on short days, oestrus and ovulation occurred 18·3 days (P < 0·05) and 23·4 days (P < 0·001) earlier than in the ewes under conditions of natural daylength. Ovulation preceded oestrus by 18·6 days and 23·1 days in Groups A and B respectively.

The occurrence of ovulation was detected by the determination of plasma progesterone concentrations. In all ewes, progesterone levels were basal until the first ovulation when the pattern of secretion was typical of that seen during the oestrous cycle. In the 4 days before ovulation, the plasma progesterone concentration increased slowly to reach a maximum of 0·66 ± 0·12 (s.e.m.) ng/ml on day − 1. The first ovulation was associated with a substantial surge of LH. Similar release of LH, thought to be related to the increased progesterone secretion, was also observed on day −5. Sporadic release of LH was also found before this time in some animals.

Plasma concentrations of FSH fluctuated randomly throughout anoestrus and during the transition to established oestrous cycles. These changes were not apparently related to ovulation. Throughout anoestrus, prolactin concentrations were raised but always declined before the time of ovulation. The fall in prolactin concentrations occurred sooner in the ewes on short days, pointing to a relationship with the decreased daylength.

These data suggest that the return to oestrous cycles may be brought about by the removal of an antigonadotrophic effect exerted by high concentrations of prolactin in the blood during anoestrus.

SUMMARY

A highly specific and sensitive heterologous double antibody radioimmunoassay for ovine follicle-stimulating hormone (oFSH) is described in detail. The assay using a rabbit antiserum to human FSH and either ¹²⁵I-labelled rat FSH or ¹²⁵I-labelled oFSH as tracer is specific for FSH. A maximum cross-reaction (B/Bo = 50%) of 0·1% was observed with other ovine, rat or human pituitary hormones or human chorionic gonadotrophin. Serum levels of oFSH in samples collected daily throughout the oestrous cycle showed large individual variations. In five out of nine animals a peak of FSH was observed on the day of oestrus.

We have shown previously that, in sheep primary pituitary cells, bone morphogenetic proteins (BMP)-4 inhibits FSHβ mRNA expression and FSH release. In contrast, in mouse LβT2 gonadotrophs, others have shown a stimulatory effect of BMPs on basal or activin-stimulated FSHβ promoter-driven transcription. As a species comparison with our previous results, we used LβT2 cells to investigate the effects of BMP-4 on gonadotrophin mRNA and secretion modulated by activin and GnRH. BMP-4 alone had no effect on FSH production, but enhanced the activin+GnRH-induced stimulation of FSHβ mRNA and FSH secretion, without any effect on follistatin mRNA. BMP-4 reduced LHβ mRNA up-regulation in response to GnRH (±activin) and decreased GnRH receptor expression, which would favour FSH, rather than LH, synthesis and secretion. In contrast to sheep pituitary gonadotrophs, which express only BMP receptor types IA (BMPRIA) and II (BMPRII), LβT2 cells also express BMPRIB. Smad1/5 phosphorylation induced by BMP-4, indicating activation of BMP signalling, was the same whether BMP-4 was used alone or combined with activin±GnRH. We hypothesized that activin and/or GnRH pathways may be modulated by BMP-4, but neither the activin-stimulated phosphorylation of Smad2/3 nor the GnRH-induced ERK1/2 or cAMP response element-binding phosphorylation were modified. However, the GnRH-induced activation of p38 MAPK was decreased by BMP-4. This was associated with increased FSHβ mRNA levels and FSH secretion, but decreased LHβ mRNA levels. These results confirm 1. BMPs as important modulators of activin and/or GnRH-stimulated gonadotrophin synthesis and release and 2. important species differences in these effects, which could relate to differences in BMP receptor expression in gonadotrophs.

Treatment of rats with bromocriptine between days 5 and 8 after the post-partum mating resulted in suppression of serum prolactin levels and caused luteal regression. Although this treatment led to embryonic resorption when suckling was prevented by removing litters soon after birth, the diapausing embryos of animals nursing a litter of eight pups were unaffected by the treatment. These results suggest that the high levels of prolactin and progesterone in the circulation during lactation are not responsible for maintenance of the diapausing state.

ABSTRACT

Active immunization of sheep against androstenedione results in an increase in ovulation rate that is associated with increased plasma levels of LH and progesterone, but not FSH. Although immunized ewes have more activated follicles the secretion rate of oestradiol is not increased. An experiment was conducted to examine the effect of androstenedione immunity on the ovarian secretion and peripheral plasma concentrations of inhibin.

Merino ewes in which the left ovary had been autotransplanted to a site in the neck were divided into control (n = 5) and androstenedione-immune (n = 6) groups. Ovarian and jugular venous blood was collected every 10 min at two stages of the follicular phase, 21–27 h and 38–42 h after a luteolytic dose of an analogue of prostaglandin F_2α (PG), and every 15 min for 6 h on day 10 of the subsequent luteal phase. The ewes were monitored regularly for luteal function by measurement of the concentration of progesterone and preovulatory LH surges. The concentration of inhibin in jugular and ovarian venous plasma was determined by radioimmunoassay and ovarian secretion rates and peripheral concentrations are expressed as pg of 1–26 peptide fragment of the α chain.

The ovarian secretion rate of inhibin tended to be greater in androstenedione-immune ewes at all stages of the oestrous cycle measured, with this difference being statistically significant (P <0·05) during the luteal phase (100±40 and 260±80 (s.e.m.) pg/min for control and immune groups respectively). The pattern of ovarian inhibin secretion exhibited pulsatile-like fluctuations which were not associated with LH pulses. Peripheral concentrations of inhibin were generally higher in immunized than in control ewes with this difference being significant (P < 0·01) from day 4 to 14 of the luteal phase (59±5 and 110±7 ng/1 for control and immune respectively). The ovarian secretion rate of immunoactive inhibin was greater (P <0·01) during the follicular phase than during the luteal phase in both groups of ewes, and peripheral concentrations of inhibin increased (P < 0·001) following injection of PG in ewes from both treatment groups.

We concluded that androstenedione immunity results in an increase in ovarian inhibin secretion, an effect that can probably be attributed to the greater number of large oestrogenic follicles present in the ovaries of these ewes. Furthermore, this increase in the concentration of inhibin may override any decrease in the negative feedback effects of ovarian steroid produced by immunization and, hence, explain the paradoxical findings of normal concentrations of FSH and raised concentrations of LH in ewes which are immunized against androstenedione.

Journal of Endocrinology (1990) 127, 285–296

SUMMARY

Oxytocin levels in pregnant and parturient guinea-pigs were studied by means of a sensitive and specific radioimmunoassay. Oxytocin was released from the maternal pituitary in substantial amount only during the expulsive phase of labour, when the mean concentration in carotid arterial blood in five animals was 503 pg/ml plasma (range 96–2900 pg/ml). Oxytocin was not found in the plasma of the first born at the moment of birth, but was usually detected in amounts ranging from 96 to 455 pg/ml in those born subsequently. The mean half-time of oxytocin in the maternal circulation during late pregnancy was 62 ± 7·5 (s.e.m., n = 5) s. In-vivo experiments showed that the placenta was permeable to oxytocin in both directions.

SUMMARY

A method is described for the extraction and concentration of oxytocin from plasma which is simpler and more rapid than other available procedures. It has proved satisfactory for both radioimmunoassay and bioassay of circulating oxytocin. The recovery of added oxytocin was 60 ± 5·5% and showed no significant variation between plasma from pregnant and non-pregnant women, or plasma from other species. The sensitivity of the assay is related to the volume of plasma extracted. With a 10 ml plasma sample and the radioimmunoassay method described previously, the maximum sensitivity under optimal conditions is 0·75 μu. (1·5pg)/ml. In the second stage of human labour, oxytocin is not detectable in maternal plasma at this level of sensitivity. Of 36 cord venous plasma samples studied, 15 showed positive results in the range 1·5–20 μu. (3–40 pg)/ml; of 16 simultaneous cord arterial and venous plasmas, 12 showed positive results; the arterial samples showed a range of 8–145 μu. (7–290 pg)/ml with an average of 45 μu./ml; the venous samples showed a range of 0–100 μu. (0–200 pg)/ml with an average of 24 μu./ml. Plasma oxytocin levels during the second stage of labour in the goat averaged 120 μu. (240 pg)/ml by radioimmunoassay and 100 μu. (200 pg)/ml by bioassay. The half-life of infused oxytocin in the non-pregnant human subject as determined by radioimmunoassay was 5 min.

ABSTRACT

The localization of inhibin α-subunit within the human corpus luteum was investigated. The antiserum used was raised in sheep against the first 1–23 amino acid sequence of the N-terminus of the human inhibin α-subunit. Using the avidin-biotin immunoperoxidase technique, intense immunostaining was localized within the granulosa-lutein cells of the corpus luteum, with absence of staining in the theca-lutein cells and surrounding ovarian tissue. Similar distribution of inhibin α-subunit immunostaining was observed in 12 corpora lutea obtained during the early, mid- and late-luteal phases and no changes in intensity were apparent at these different stages. Negative controls were obtained by applying antiserum which had been preabsorbed overnight with excess inhibin peptide in place of primary antiserum and also normal non-immune sheep serum as a substitute for primary antiserum. These results provide further evidence that the human corpus luteum is a significant source of immunoreactive inhibin during the normal human menstrual cycle. The specific localization within the granulosalutein cells of the corpus luteum suggests that inhibin α-subunit production may originate from a discrete cell population within the human corpus luteum.

Journal of Endocrinology (1991) 129, 155–160