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M. R. Luck
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The physiological role of ovarian oxytocin has been much debated since its (re)discovery at the beginning of the decade (Wathes, 1984). As a neuropeptide secreted in abundance from a non-neural tissue, ovarian oxytocin has been a good example of the 'ectopic' endocrinology discussed by Henderson (1987). As pointed out recently (Auletta, Jones & Flint, 1988), we are still only certain of its secretion in major quantities in domestic ruminants. For these species, much experimental evidence has accumulated at both the animal and tissue levels regarding the circumstances and mechanism of its secretion. The problem has been to define exactly what role it may play in the endocrinology of the reproductive cycle. Neither of the two main hypotheses put forward to account for the presence of oxytocin in the ovary has proved to be entirely satisfactory.

The first hypothesis proposes a paracrine role for oxytocin in the regulation of ovarian steroidogenesis.

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M. R. Luck
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B. Jungclas
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ABSTRACT

The effects of catecholamines and ascorbic acid on cultured bovine granulosa cells have been examined to assess their possible role in the initiation and maintenance of luteal oxytocin secretion. The actions of these agents have also been compared with the previously reported ability of follicular theca tissue to enhance oxytocin secretion.

Using granulosa cells cultured in serum-supplemented medium, we observed a highly significant enhancement of oxytocin secretion in the presence of adrenaline and noradrenaline, particularly over the concentration range 1–10 μmol/l. This effect was accompanied by smaller and less consistent changes in progesterone secretion and did not involve any change in the time-course of oxytocin secretion. Acetylcholine was without effect. Ascorbic acid stimulated oxytocin secretion when used alone over a range of concentrations, but was also able to synergize with adrenaline. Lactic acid was ineffective. The stimulation of oxytocin secretion by adrenaline could be blocked by equimolar propranolol, but the stimulation of progesterone was not blocked. Propranolol had a variable effect on the ability of theca tissue to stimulate oxytocin secretion by granulosa cells but the results also suggested the presence of some β-agonistic activity in the culture medium.

We conclude, first, that catecholamines may be involved in the regulation of ovarian oxytocin secretion, secondly, that ascorbate may regulate oxytocin secretion through its involvement in the biosynthesis of oxytocin but also through interaction with catecholamines and, thirdly, that the stimulatory action of theca tissue probably does not involve the action of β-agonists.

J. Endocr. (1987) 114, 423–430

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M. R. Luck
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B. Jungclas
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ABSTRACT

Bovine granulosa cells secrete oxytocin when cultured in a serum-supplemented medium. The time-course of secretion is similar to that in the early corpus luteum in vivo, with a delay of 1 to 2 days followed by a peak and decline over the first 5 days of culture. We have investigated the basis of this time-course in vitro and studied the temporal characteristics of the stimulatory actions of ascorbic acid and adrenaline on this process.

Cells cultured on stirred microcarriers showed a similar pattern of secretion of oxytocin to those cultured on conventional flat plates, despite continuing and rapid mitosis. This indicated that the secretion profile in conventional culture was not an artifact related to the cessation of mitosis. Furthermore, secretion of oxytocin and progesterone by cells on microcarriers was stimulated without a corresponding change in mitotic rate, showing that the secretion per cell had been increased.

In conventional culture, addition of ascorbic acid to culture media (0·5 mmol/l) increased the secretion of oxytocin (up to 4·5-fold) but only if ascorbic acid was present during the first day of culture. The cells showed a progressive refractoriness to stimulation after 12 h. Since the time-course of secretion was unaltered by treatment, this resulted in a delay of 1 to 2 days before the action of the ascorbate was seen. The secretion of progesterone was similarly affected but with less stimulation and less consistency. In contrast, cells treated with adrenaline (10 μmol/l) secreted more oxytocin on the day of treatment and did so at any time during culture provided that there was sufficient basal secretion of hormone. Adrenaline also failed to alter the time-course of secretion but treated cells showed a persistent response, maintaining enhanced secretion for up to 3 days after the adrenaline had been removed.

Ascorbate and adrenaline were highly synergistic in their effects, provided that the ascorbate was present from the start of culture; the response to adrenaline strongly reflected the degree of ascorbate stimulation. We conclude that granulosa cells secrete oxytocin according to an inherent time-schedule and that there is a limited period during which they can respond to ascorbate. Since ascorbate is required for the biosynthesis of oxytocin, this suggests that the availability of ascorbate during corpus luteum formation may determine the amount of oxytocin which can be released subsequently in response to catecholamines.

J. Endocr. (1988) 116, 247–258

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B. Jungclas
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M.R. Luck
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ABSTRACT

We have examined the ability of granulosa cells, from carefully selected preovulatory bovine follicles, to secrete oxytocin in vitro. Although cells from 83% of follicles underwent functional luteinisation (greatly increased progesterone secretion) in serum-supplemented culture, only 69% had cells capable of oxytocin secretion. Secretion followed a similar time course in all cultures, with the peak appearing on day 3. Oxytocin, but not progesterone, output could be consistently increased by addition of pieces of theca interna tissue, or theca conditioned medium, to the cultures. The effect could be achieved by exposure to theca tissue at any time prior to peak output without altering the time course of secretion. Oxytocin could not be detected in follicular fluid from any of the selected follicles, nor in medium from theca cultured alone. We conclude that the potential for oxytocin secretion is a feature of follicular maturation which is lost during atresia and that the stimulus to secretion is associated with luteinisation but not with progesterone output. Finally, the intermixing of follicular cells during corpus luteum formation may provide a mechanism for the enhancement of oxytocin secretion within a predetermined time frame.

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M R Luck
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Y Zhao
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Introduction

A unique feature of reproductive tissues is their state of continual change. All tissues undergo differentiation and development during embryology, but the reproductive organs have cycles of tissue activity as a characteristic and integral feature of their mature, functional state. These cycles may operate over long or short periods of time (Fig. 1). For example, the mammary gland may have a period of secretory activity lasting several years, preceded and followed by states of extended but reversible quiescence. In contrast, the ovary may show cycles of follicle growth, ovulation and rupture repeated over as short a period as 7 days (Didelphis marsupialis; Hayssen et al. 1993). Between these extremes are cycles of variable length in tissues such as the uterus (proliferation, deciduation, pregnancy, parturition) or the seasonal testis (growth, spermatogenesis, regression). All these cycles are superimposed on the developmental changes associated with maturation and senescence to which all parts

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M.R. Luck
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J.A Shale
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J.H. Payne
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ABSTRACT

The ruminant conceptus secretes proteins during early pregnancy which maintain the corpus luteum. These trophoblast proteins are related to the αII-interferons and prevent luteolysis indirectly by disrupting the secretion of endometrial prostaglandin. Although trophoblast interferons appear to be largely confined to the uterine lumen, it remains possible that they also act peripherally. This report describes in vitro studies which suggest that interferon may influence hormone secretion by the ovary directly. The study employed i) a well defined serum-free culture model in which bovine granulosa cells secrete the luteal hormones progesterone and oxytocin, and ii) serum-free and serum-supplemented cultures of cells from early CL. Dose-response experiments were performed using bovine recombinant α-interferon (brIFN). Progesterone and oxytocin secretions were measured over 4-5 days of culture and DNA content was also determined.

Low concentrations of brIFN (10−15 mol/l to 10−11 mol/l) stimulated progesterone secretion by granulosa cells by up to three fold, without significantly affecting oxytocin concentrations or culture DNA content. Concentrations of 10−10 mol/l to 10−7 mol/l suppressed progesterone secretion in a log dose-related manner (r=0.97) with evidence of toxicity (lower oxytocin concentrations and significantly reduced DNA compared with controls). Progesterone secretion by luteal cells in serum-free culture was stimulated in the presence of 10−15 mol/l brIFN, whilst high concentrations again caused inhibition. The data show that ovarian cells can respond directly to low concentrations of interferon-like proteins. They also demonstrate an inhibition by high doses which may mask the stimulatory effect in this model. The data suggest that the early corpus luteum may be directly influenced by α-interferon. A stimulation of progesterone, but not of oxytocin, secretion from ovarian cells would be consistent with a role for conceptus proteins in maintaining the corpus luteum of pregnancy.

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