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Stricker & Gruether (1928, 1929) were the first to observe that whole pituitary extracts could initiate lactation in pseudopregnant ovariectomized rabbits. Greatly assisted by the many advantages of the specific pigeon crop sac response for isolation and quantitative assay of the effective protein, Riddle, Bates & Dykshorn (1933) isolated it and named it 'prolactin'. Since this early work, prolactin has been shown to have an important luteotrophic rôle in many species. In this rôle it is usually associated with LH.In studying the part played by prolactin in reproductive processes, we have investigated its secretion throughout the oestrous cycle and during pregnancy in the sheep, and have studied the effects of replacement therapy in hypophysectomized and pituitary stalk-sectioned ewes.All the experiments have been carried out on ewes of

THE corpus luteum is an endocrine gland which plays an active, yet transitory role in female mammals after puberty. During every luteal phase, the length of which varies with the species, hormones influence the formation, growth, secretory activity, and degeneration of the gland. Some hormones cause cellular differentiation and multiplication, phenomena which lead us to think that these substances affect nucleic acids. In a brief interval of a few days events occur, which, in other organs may be spread out over a lifetime. This fact draws our attention to the complexity of the rather short-term hormonal regulation which influences luteal cells.Research done during the last 10 yr., mostly on domestic animals, indicates that there are two hormonal systems affecting the corpus luteum. The first ensures morphogenesis and induces or stimulates progesterone secretion, whereas the second causes rapid stoppage of secretory phenomena and morphological regression. Luteolytic effects, principally, but not exclusively, of the uterus, have been the subject of a report by Dr. Melampy and will not be discussed here.

Experiments were carried out with a view to determining the relative importance of the pituitary gland and the uterus for the luteolytic action of oestradiol in the cycling ewe.The influence of the pituitary was modified by stalk section or hypophysectomy and that of the uterus by hysterectomy. Oestradiol (0.5 mg twice daily) caused an increase in ghe life-span of the corpus luteum (CL) if treatment began on day 3 of the cycle, and a decrease when injections commenced on days 9 or 11.Pituitary stalk sections performed on days 3 or 11 caused little change in CL weight on day 14. However, in either case, oestradiol administration from day 11 resulted in a large fall in CL weight, indicating that intact pituitary connections to the hypothalamus were not essential for the luteolytic action of oestradiol. Luteal regression following hypophysectomy on day 11 was accelerated by oestradiol benzoate injected from day 11. On the other hand, hysterectomy on day 11 always resulted in maintenance of the CL to day 20, whether or not stalk section or oestradiol administration had taken place. This indicated that the presence of the uterus was essential for the luteolytic effects of oestradiol.These experiments show that the luteolytic properties of oestradiol reflect mainly a direct action of this hormone on the uterus.

Oestradiol has been shown to cause either premature luteal regression or prolonged luteal maintenance depending on whether it is administered early or late in the oestrous cycle. Although these effects are not observable in the absence of the uterus, it was considered desirable to examine the possibility that the pituitary gland as well as the uterus may be involved in this phenomenon. Accordingly, plasma LH and prolactin concentrations were studied in sheep treated with oestradiol from day 3 (luteotrophic action) or day 11 (luteolytic action) with and without pituitary stalk section. Plasma LH fell below the limit of detection (0.3 ng/ml) in the plasma of sheep after pituitary stalk section, whether or not oestradiol had been administered. In the intact ewe, oestradiol given from day 3 resulted in luteal maintenance associated with a significant increase in the secretion of plasma LH and prolactin. However, similar treatment in the stalk sectioned ewe also resulted in luteal maintenance without any major increase in gonadotrophin secretion. On the other hand, when oestradiol was given from day 11, there was no evidence either from the intact or the stalk sectioned sheep that the pituitary contributed to the luteolytic action of oestradiol may possibly have a pituitary component, the luteolytic action is probably mediated solely by the oestrogenic action on the uterus.

The milk-ejection reflex after different lesions of the sensory system was studied by mechanical milking of the ewe. Only animals with lesions which did not produce locomotor ataxia were investigated. Section of the dorsal tract of the spinal cord at different thoracic levels blocked the milk-ejection reflex. This was not due to postoperative shock, because the same type of lesion made at the sacral level was without effect. Moreover, unilateral section of this tract blocked the reflex when milking was limited to the ipsilateral mammary gland. At the cervical level, the interruption of the dorsal tract was ineffective, which can probably be explained by the existence, in sheep, of the spino-cervico-thalamic tract. The latter becomes ventral at the cervical level. Lesions of the lemniscal system in the mesencephalon (bilateral coagulation of the medial lemniscus) and in the thalamus (bilateral destruction of the ventral posterolateral nucleus) do not inhibit the milk-ejection reflex.The role of the spinal lemniscal system is discussed in relation to the work of others and to the electrophysiological results obtained by one of us. It is suggested that two sensory systems (lemniscal and extralemniscal) have to interact to induce the release of oxytocin evoked by the stimuli of mammary origin during milking.

Progesterone injected daily at different stages of pregnancy in the rabbit, reduces or temporarily blocks mammary secretion measured on the 24th day either by the incorporation of [14C] glucose into lactose or by the [12C] lactose content of mammary tissues. Progesterone does not affect the number of mammary cells, as determined by the DNA content of the mammary gland, while the quantity of RNA is only reduced if the hormone is injected from the beginning of pregnancy. These results suggest that progesterone has an inhibitory effect on secretory activity, which mainly takes place during the first two thirds of pregnancy.From day 19, twice daily administration of 12.5 IU of prolactin stimulates milk secretion and increases the content of nucleic acids measured on the 24th day. Progesterone (5 mg twice daily) does not prevent these lactogenic effects, nor does it modify the increase in uncleic acids caused by prolactin treatment. Despite the continued presence of progesterone, prolactin has the same qualitative properties in pregnant rabbits deprived of their gonads or pituitary as it has in intact rabbits. Hence prolactin plays an essential role in the mammogenesis and lactogenesis occurring during pregnancy in the rabbit.In addition, our studies show that ovarian secretions other than progesterone are effective under normal conditions of pregnancy, since the administration of progesterone in ovariectomized pregnant rabbit does not allow normal mammary growth despite the maintenance of pregnancy.

The rate of biosynthesis of lactose from [14C]glucose was used to study the action of different hormone combinations on cultures of mammary explants from pseudopregnant rabbits. Insulin with or without cortisol had no effect, but insulin with prolactin induced lactose synthesis in vitroeven when prolactin was not added to the culture medium until 48 h after culture began. Insulin together with cortisol and prolactin produced a greater response when used from the beginning of culture. Differences between these results and those obtained with mammary tissue from pregnant mice are discussed.

Twice-daily intramuscular injections of 0·5 mg oestradiol benzoate, starting on Day 3 of the cycle, were able to prolong the life of the corpora lutea in sheep, as judged by their weight, their DNA and RNA content, and the progesterone concentration in ovarian vein blood. This luteotrophic effect persisted even when the pituitary stalks of the animals were sectioned, but it was abolished when the animals were hypophysectomized.Four possible sites of oestrogen action were considered; the hypothalamus, the pituitary, the corpus luteum and the uterus. It was concluded that oestrogen is luteotrophic principally because of its action on the uterus, where it seems to interfere with the normal luteolytic mechanism.

The purpose of these experiments was to investigate the part played by the pituitary gland and the uterus in the control of the corpus luteum in the sheep. Six experiments were carried out, as follows:Hypophysectomy early and late in the oestrous cycle. Hypophysectomy on days 2–5 allowed the corpus luteum to continue secreting normal amounts of progesterone for up to 9 days, but thereafter the secretion declined. Hypophysectomy on days 9–10 caused the progesterone secretion to fall within 4 days. Thus it seems that the corpus luteum has a limited functional life span, and it normally requires the presence of a pituitary luteotrophin during the second half of the oestrous cycle if it is to secrete normally for 15 days.Pituitary stalk section early and late in the oestrous cycle. Regardless of the time of stalk section, the corpus luteum behaves as it would during a normal cycle. Thus pituitary stalk section does not produce the same effect as hypophysectomy, and seems to allow the severed pituitary gland to continue secreting luteotrophin.Hypophysectomy and hysterectomy carried out simultaneously early in the cycle. The results were similar to those in 1, progesterone secretion having declined significantly by days 12–15. This confirms that the declining secretory activity is due to a deficiency of pituitary luteotrophin, and not to any uterine luteolytic effect in this experiment.Pituitary stalk section and hysterectomy carried out simultaneously early in the cycle. In striking contrast to 3, some corpora lutea were stillsecreting progesterone normally on day 18. This shows once again that the isolated pituitary gland can continue to secrete luteotrophin. The different responses in this experiment and 2 emphasise the fact that the uterine luteolytic effect is normally dominant to the pituitary luteotrophic stimulus. Thus it would be impossible to demonstrate luteotrophic activity if the uterus were still present.Hysterectomy carried out at mid cycle, followed by hypophysectomy 20–30 days later. Whilst hysterectomy alone prolongs the secretory activity of the corpus luteum, subsequent hypophysectomy results in a rapid decline in progesterone secretion, commencing 48 hours after the operation. Thus the corpora lutea prolonged by hysterectomy, unlike those of the normal cycle, require daily pituitary luteotrophin secretion for their continued existence.Hysterectomy carried out at mid cycle, followed by pituitary stalk section 20–30 days later. Unlike 5, stalk section allows the corpora lutea to continue to secrete progesterone in large amounts for at least 15 days after the operation. This experiment, together with 2 and 4, once again emphasises that the stalk-sectioned pituitary gland can continue to secrete luteotrophin, at least for a time.These experiments therefore support the view that the cyclical corpus luteum of the sheep is under a dual control. There is a pituitary luteotrophin, whose secretion continues after stalk section, and a uterine luteolysin that is dominant to the luteotrophic stimulus, and can still function normally after pituitary stalk section.The fact that the corpus luteum of the hysterectomised animal cannot function for more than about 15 days after stalk section suggests that the luteotrophic stimulus may be complex, possibly envolving more than one hormone.

The functional activity of ovine CL was assessed by their weight, DNA and RNA content, and the concentration of progesterone in ovarian venous blood.If sheep were hysterectomized on Days 9 to 12 of the oestrous cycle, the CL were maintained in a fully functional state until at least Day 60, but their activity had begun to decline by Day 128 to 135. When hysterectomized sheep were hypophysectomized, there was a significant decline in luteal activity within 48 hr, regardless of whether or not the pituitary stalk and pars tuberalis were left intact. The CL had almost completely stopped secreting progesterone within 4 days of hypophysectomy.Hysterectomized, hypophysectomized animals were therefore used in a series of experiments to test the luteotrophic properties of sheep pituitary gonadotrophins. Doses of up to 5 mg FSH/day were unable to prevent complete luteal regression; similarly, doses of up to 5 mg LH/ day were also without effect. When mixtures of FSH and LH were given, the results were no better. However, prolactin in doses of up to 1000 i.u./ day was invariably able to maintain functional CL for 12 days, although at a considerably reduced level of activity. When prolactin was combined with a small dose of LH (0·25 mg/day), the CL were maintained at a level of activity comparable to that seen in hysterectomized animals before hypophysectomy. The slight synergistic action of FSH with prolactin was probably due to LH contamination. No further beneficial effects were obtained by adding FSH to the prolactin—LH mixture.We conclude that prolactin and LH are both necessary for the maintenance of the ovine CL, and that these two hormones together make up the `luteotrophic complex'. But whilst prolactin on its own has some luteotrophic activity, LH by itself is completely ineffective.