Sow Corpora Lutea Research Articles

Evidence for a switch in the site of relaxin production from small theca-derived cells to large luteal cells during early pregnancy in the pig.

The presence of immunoreactive relaxin was studied in corpora lutea of sows during the oestrous cycle and early pregnancy by immunohistochemistry and radioimmunoassay using three different anti-relaxin sera. Sections were immunostained using the peroxidase-anti-peroxidase or the immunogold-silver technique. Before Day 14, staining in corpora lutea from non-pregnant and pregnant animals was indistinguishable. With all antisera, no immunostaining was seen on Day 3, but was detected on Days 5-7 in cells from the theca interna. In non-pregnant animals, this immunostaining decreased and by Day 15 only an occasional large cell in the centre of the corpus luteum was stained. No staining was seen by Day 22. The relaxin content of corpora lutea measured by radioimmunoassay remained low throughout the luteal phase. In contrast, the amount of immunoreactive relaxin in corpora lutea rose dramatically (140-fold) between Days 11 and 14 of pregnancy and by Day 14 of pregnancy immunostaining was seen in the majority of large luteal cells. By Day 20 of pregnancy the concentrations of immunoreactive relaxin had further increased. Histochemical staining for alkaline phosphatase suggested that, while the relaxin-immunoreactive cells seen in the early luteal phase may be theca-derived, those during early pregnancy may be derived from the granulosa. The results are compatible with the suggestion that relaxin is produced by theca-derived cells during the early luteal phase and that between Days 11 and 14 there is a switch in the site of relaxin synthesis from theca-derived cells to granulosa-derived large luteal cells. In the absence of luteolysis, as during pregnancy, this switch is accompanied by a dramatic increase in relaxin synthesis.

Immunocytochemical Localization of Relaxin in Corpora Lutea of Sows Throughout the Estrous Cycle1

Porcine relaxin has been sought by localization in the corpus luteum of sows on Days 3, 7, 9, 11, 12, 15, 18, 19, and 21 of the estrous cycle, using the avidin-biotin immunoperoxidase method and an antiserum to purified porcine relaxin. Simultaneous localization of relaxin in corpora lutea from sows on Days 108 and 113 of pregnancy was used to compare the intensity of immunostaining with that of corpora lutea of cyclic animals. However, the antiserum dilution necessary for optimal localization differed considerably in these two states (1:10,000 in pregnancy and 1:750 in the cycle), suggesting that lower levels of antigen are present in the luteal cells of the cycle. Relaxin immunostaining was undetectable on Day 3 of the cycle but became evident by Days 7 and 9. At Day 11 staining intensity increased and persisted through Day 15. On Day 18 some stain was still evident, but by Days 19, 20, and 21 there was complete absence of immunostain. Relaxin immunostaining appeared to be located throughout the cytoplasm of the luteal cell, as clear areas in the nuclear region were often observed. The results suggest that relaxin is produced in low amounts by the luteal cells of the cyclic sow and that the levels fluctuate with stage of the cycle. Lack of evidence from radioimmunoassay for a surge of relaxin secretion into the systemic circulation prior to luteolysis in the pig estrous cycle suggests that the relaxin localized in the luteal cells of the cycle may have an intraovarian function.

Bioassay methods for relaxin: uses and pitfalls.

The ability of aqueous extracts of porcine corpora lutea of pregnancy to “relax” the pubic symphysis of the estrogen-primed ovariectomized guinea pig was first described by Hisaw (1926). Subsequent work showed that porcine luteal extracts which contained guinea pig pubic symphysis-relaxing activity could inhibit contractions of uterine myometrium of rats in vitro (Sawyer et al. 1953) and guinea pigs in vivo (Krantz et al.3 1950). Around the same time, it was reported that crude extracts of sow corpora lutea induced interpubic ligament formation in estrogen primed mice (Hall and Newton, 1946; Kliman et al. 3 1953). These basic observations led to the development of numerous bioassay methods for relaxin which have been amply documented in previous reviews (Frieden and Hisaw, 1953; Hall, 1960; Steinetz et al. 1969). The present paper will, therefore, be devoted primarily to a discussion of the uses (and abuses!) of the various assay methods, the pitfalls which may be encountered in assaying relaxin activity in impure extracts and the growing realization that relaxin activity may be associated with a family of related polypeptides. Of special interest are recent observations which suggest that relaxin-like hormones obtained from different species may not be equally effective in the classical bioassay methods.

Isolation and characterization of porcine and rat relaxin.

Hisaw (1926) first reported that experimentally induced “relaxation” of the pubic ligament in guinea pigs was controlled by hormones. In 1930 Fevold, Hisaw, and Meyer (1930) showed that an aqueous extract of sow corpora lutea caused relaxation of the pubic ligament in estrogen-primed, ovariectomized guinea pigs. They named the active substance relaxin. Essentially all subsequent efforts to isolate relaxin employed ovaries obtained from pigs daring late pregnancy since this source of relaxin is known to have a high content of relaxin activity and is relatively easy to acquire. Progress toward the isolation of porcine relaxin was slow. It was not until the 1950’s and early 1960’s when new techniques for the isolation and characterization of proteins, as well as improved methods for relaxin bioassay (Steinetz et al.3 1960, 1969), became available that well documented advancement was made in relaxin purification efforts.

Primary structure of porcine relaxin: homology with insulin and related growth factors

HISAW and co-workers1,2 first described a factor from sow corpora lutea that caused relaxation of the pubic symphysis in oestrogen-primed guinea pigs. Subsequent studies have shown that the active substance, termed relaxin, is a low molecular weight peptide hormone. Its presence has been demonstrated during pregnancy in female pigs, guinea pigs, rabbits, mice and rats3,4, and, more recently, in humans5. Its actions include dilatation and softening of the cervix, inhibition of uterine contractions and relaxation of the pubic symphysis and other pelvic joints. Relaxin, which has a molecular weight of approximately 6,000, consists of two non-identical chains joined by disulphide bridges6. We have now determined, and report here, the complete amino acid sequence of porcine relaxin.

Qualitative and quantitative studies on the innervation of the corpus luteum of rat and pig

Qualitative and quantitative studies were made to determine the amount of nerve fiber supplying corpora lutea (CL) of rats during the oestrous cycle and pregnancy and sow CL during days 4–6 after ovulation. Fluorescence microscopy of freeze-dried, paraformaldehyde treated (Falck-Hillarp method) rat ovaries reveals adrenergic nerve fibers which run along with vessels and form a network among interstitial gland cells. Nerve fibers do not enter the granulosa cell layer in follicles or CL. In the CL circumference both vascular and non-vascular nerves occur the latter being related to the fibromuscular layer and probably innervating smooth muscle cells. No striking differences exist between the innervation of the ovary in non-pregnant and pregnant rats. Bodian and methylene blue staining did not contribute to a more detailed knowledge of rat ovary nerve supply. Electron microscopic quantitative analysis of rat and pig CL (rat: day 18 of pregnancy; pig: day 4–6 after ovulation) revealed no axon profiles in 2.000 grid squares (one square measuring 2.25×10-2 mm2) of randomly taken CL sections. Thus it was possible to calculate an upper limit of 133 μm of nerve fibers per 1 mm3 CL tissue, in case there were any at all.

The fine structure of sow lutein cells.

AbstractThe morphological observations described in this report are part of a biochemical study of progesterone intermediates using sow ovary. This tissue is extensively involved in the production of some steroid hormones. Because the lutein cells are thought to be involved in the synthesis of progesterone and its intermediates and because these cells constitute by size and number the greatest portion of the mass of sow ovary, we confined the morphological investigation to these cells. Sow corpora lutea, fixed with glutaraldehyde and osmium tetroxide were examined by light and electron microscopy.The lutein cells of the sow are large epithelioid cells with oval nuclei and extensive amounts of cytoplasm. Large masses of highly organized smooth endoplasmic reticulum, many oval mitochondria with tubular cristae, and many lipid droplets are present. Only a few profiles of rough endoplasmic reticulum are seen in any one section, and Golgi membranes cannot by distinguished from other smooth membranes. The smooth endoplasmic reticulum is the major membranous organelle in these luteal cells. Microsomal preparations from luteal cells must be very rich in fragments of these smooth membranes. The demonstration of many steroidogenic enzyme systems in microsomal preparations lends strong support to the supposition that these enzyme systems reside in the smooth endoplasmic reticulum.

The Physiology of Relaxin

Publisher Summary This chapter discusses the physiology of relaxin. The discovery of relaxin was the outcome of studies concerning pelvic adaptations associated with parturition. Relaxin has been extracted from the corpora lutea of sows by several different methods. The chief chemical characteristics of relaxin, as judged by these extracts, were its isoelectric point of 5.4 to 5.5, stability in acid solutions, and its insolubility in the common nonpolar solvents. The ovary, uterus, and placenta have been suggested as possible sites of relaxin formation. Not only has it been possible to extract relaxin from these three organs, but there is indication that they may serve as sources of the hormone in the normal physiology of the animal. A normal concentration of 10 G.P. units of relaxin per ml. of blood serum is maintained in pregnant rabbits when they are castrated at midterm and abortion is prevented by giving progesterone. The formation of relaxin in the mouse is apparently different from that observed in guinea pigs and rabbits.

AN IMPROVED METHOD OF EXTRACTION AND PURIFICATION OF RELAXIN FROM FRESH WHOLE OVARIES OF THE SOW1

RELAXIN, a water-soluble, physiologically active substance of the ovary has been prepared in partly purified form by two different methods. Fevold, Hisaw, and Meyer (1932) extracted fresh sow corpora lutea with acid alcohol and worked up the active material by solution in and precipitation from organic solvents. The activity of the final product, prepared as an iso-electric precipitate at pH 5.4, was 30 units per mg. Using semi-dried, defatted luteal tissue obtained as a by-product from a commercial procedure for the preparation of progestin, Albert, Money, and Zarrow (1946) prepared relaxin with approximately the same order of activity by differential solubility in water, ammonium sulfate solutions and cold dilute alcohol.

THE EFFECT OF PROGESTIN AND PROGESTERONE ON OVULATION IN THE RABBIT

The ability of exogenous progestin derived from the corpora lutea of sows or progesterone to inhibit ovulation was studied in the rabbit. The animals received either 3 rabbit units of progestin or 1-5 mg of progesterone after which attempts at mating were made most of which were unsuccessful. In those animals in which mating was achieved there was no indication of ovulation in any of the animals 18 hours after mating. The administration of a minimal ovulating dose of pregnancy urine extract immediately after the refusal of mating resulted in ovulation in 7 of 9 animals. It was concluded that progestin inhibits ovulation by interfering with the postcoital release of pituitary gonado tropin hormone.

Isolation of the Relaxative Hormone on the Corpus Luteum.

Pelvic ligaments of guinea pigs undergo very pronounced relaxation during pregnancy to facilitate the birth of young. While studying the physiology of this reaction, one of us discovered that it was under hormonal control and that the substance responsible was present in the blood of certain mammals during pregnancy.1 This hormone is capable of producing relaxation of the pelvic ligaments of virgin guinea pigs in a fashion typical of pregnancy, if given while the animals are in or recovering from oestrum. Later it was found that this reaction could also be produced through the use of extracts of corpora lutea of sows' ovaries. General methods for the preparation of these extracts have been given elsewhere.2 These corpus luteum extracts, in addition to relaxing the pelvic ligaments, also produced other physiological changes ordinarily attributed to the corpus luteum, such as inhibition of ovulation, vacuolation of the vaginal mucosa of rats,3 production of pseudo-pregnancy in rabbits,4 and production of pr...