Corpus Luteum Extract Research Articles

Progesterone – Historical perspective

Research leading to the discovery and characterization of progesterone (P4) began in the mid 1800 s and followed a path carved by key discoveries in the burgeoning field of endocrinology. The primary observations leading to the discovery of P4 was that the maternal corpus luteum (CL) is necessary for the establishment and maintenance of pregnancy. Experiments in animal models exploring the consequence of CL ablation and the effects of treatment with CL extract soon followed and formed the basis for the eventual isolation and characterization in 1930 s of the CL hormone, initially referred to as progestin, and subsequently named progesterone. In the following decades research into the physiology, biochemistry and molecular biology of P4 in the context of pregnancy provided fundamental insights into the hormonal control of pregnancy establishment, maintenance and termination. This review highlights the work of pioneering researchers and their seminal discoveries in the research history of P4.

Time-resolved fluoroimmunoassay (TR-FIA) of porcine relaxin.

We developed and validated a new assay system for porcine relaxin that overcame the drawbacks of RIA by adapting time-resolved fluoroimmunoassay (TR-FIA), which was recently introduced as a non-RIA format. The assay system was a solid-phase TR-FIA based on competition for a polyclonal anti-porcine relaxin antibody between europium (Eu)-labeled porcine relaxin and test samples. Antibody-relaxin complexes were then bound to the second antibody coated on the solid phase, achieving rapid and complete separation of bound and free antigen. A standard curve was produced over the range of 1 pg/well to 1000 pg/well. Serum and corpus luteum extracts from pigs in late pregnancy exhibited inhibition curves parallel to that of the relaxin standard, whereas male pig serum caused no displacement of the labeled hormone. No cross-reactivity was seen with other hormones, such as insulin, LH, and FSH, indicating a high specificity of the assay. The sensitivity was 4 pg/well (80 pg/ml), which was high and equivalent to that of the porcine relaxin RIA. The intra-assay and inter-assay coefficients of variation were less than 3.8% and 6.7%, respectively. Recovery of porcine relaxin added to male pig serum sample averaged 103%. The advantages of this TR-FIA were that addition of tyrosine was not necessary for labeling, unlike the RIA, Eu-labeled relaxin was stable enough to allow long-term storage for more than one year, the assay was completed in only 5 h versus two to seven days for the RIA, and no special safety precautions were needed. To validate this TR-FIA, the serum relaxin concentrations during late pregnancy, parturition and early lactation were investigated in pigs. Serum relaxin levels determined by this assay were similar to those obtained previously by RIA. In conclusion, this TR-FIA could replace RIA as the method of choice for assay of relaxin.

Progesterone metabolites in breast cancer

In the 70 years since progesterone (P) was identified in corpus luteum extracts, its metabolism has been examined extensively in many tissues and cell lines from numerous species. In addition to the reproductive tissues and adrenals, every other tissue that has been investigated appears to have one or more P-metabolizing enzyme, each of which is specific for a particular site on the P molecule. In the past, the actions of the P metabolizing enzymes generally have been equated to a means of reducing the P concentration in the tissue microenvironment, and the products have been dismissed as inactive waste metabolites. In human breast tissues and cell lines, the following P-metabolizing enzymes have been identified: 5alpha-reductase, 3alpha-hydroxysteroid oxidoreductase (3alpha-HSO), 3beta-HSO, 20alpha-HSO, and 6alpha-hydroxylase. Rather than providing diverse pathways for inactivating and controlling the concentration of P in breast tissue microenvironments, it is proposed that the enzymes act directly on P to produce two types of autocrines/paracrines with opposing regulatory roles in breast cancer. Evidence is reviewed which shows that P is directly converted to the 4-pregnenes, 3alpha-hydroxy-4-pregnen-20-one (3alpha-dihydroprogesterone; 3alphaHP) and 20alpha-dihydroprogesterone (20alphaHP), by the actions of 3alpha-HSO and 20alpha-HSO respectively and to the 5alpha-pregnane, 5alpha-pregnane-3,20-dione(5alpha-dihydroprogesterone; 5alphaP), by the irreversible action of 5alpha-reductase. In vitro studies on a number of breast cell lines indicate that 3alphaHP promotes normalcy by downregulating cell proliferation and detachment, whereas 5alphaP promotes mitogenesis and metastasis by stimulating cell proliferation and detachment. The hormones bind to novel, separate, and specific plasma membrane-based receptors and influence opposing actions on mitosis, apoptosis, and cytoskeletal and adhesion plaque molecules via cell signaling pathways. In normal tissue, the ratio of 4-pregnenes:5alpha-pregnanes is high because of high P 3alpha- and 20alpha-HSO activities/expression and low P 5alpha-reductase activity/expression. In breast tumor tissue and tumorigenic cell lines, the ratio is reversed in favor of the 5alpha-pregnanes because of altered P-metabolizing enzyme activities/expression. The evidence suggests that the promotion of breast cancer is related to changes in in situ concentrations of cancer-inhibiting and -promoting P metabolites. Current estrogen-based theories and therapies apply to only a fraction of all breast cancers; the majority (about two-thirds) of breast cancer cases are estrogen-insensitive and have lacked endocrine explanations. As the P metabolites, 5alphaP and 3alphaHP, have been shown to act with equal efficacy on all breast cell lines tested, regardless of their tumorigenicity, estrogen sensitivity, and estrogen receptor/progesterone receptor status, it is proposed that they offer a new hormonal basis for all forms of breast cancer. New diagnostic and therapeutic possibilities for breast cancer progression, control, regression, and prevention are suggested.

Physiology of the corpus luteum

My life with progesteroneAmerican Journal of Obstetrics & GynecologyVol. 193Issue 4PreviewThe twists and turns of fate have linked my name so many times with the other ovarian hormone that I still shudder when some senior citizen says, “You must have been awfully young when you worked with Doisy.” The confusion arises because there are 2 Allens whose names have become attached to the 2 ovarian hormones. Edgar Allen (1892-1943) and Edward Adelbert Doisy (1893-1986) announced their discovery of a hormone in the ovarian follicular fluid in 1923.1 George Washington Corner (1889-1981) and I announced the discovery of a second ovarian hormone in the corpus luteum in 1929. Full-Text PDF Comment by Lawrence D. Longo, MDAmerican Journal of Obstetrics & GynecologyVol. 193Issue 4PreviewGeorge Washington Corner (1889-1981) and Willard Myron Allen (1904-1993) (the latter a medical student at the time) first obtained a secretory endometrium in the rabbit uterus after injection of an extract of corpus luteum, which laid the groundwork for the isolation of progesterone. Illustrated above, several figures are from a nonpregnant rabbit uterus after an ophorectomy and various treatments: 1 and 5, Normal control from mature rabbit; 7, control from immature rabbit; 2, proliferative endometrium in response to 5 days treatment with corpus luteum extract; 3, same after a larger dose; 8, same in immature animal; 4, same after one-half minimal effective dose; 6, treatment with follicular fluid (presumably containing estrogen); 9, same in immature animal. Full-Text PDF

Demonstration of a non-steroidal, non-inhibin factor in the ovine corpus luteum of pregnancy that reduces pituitary responsiveness to GnRH-induced LH secretion in vitro.

The decline in pulsatile LH secretion and pituitary responsiveness to GnRH as pregnancy advances may be due to non-steroidal factors secreted by the ovine corpus luteum of pregnancy. Corpora lutea from ten ewes on days 70-80 of gestation were homogenized, charcoal-treated and, together with charcoal-treated follicular fluid from superovulated women, were subjected to inhibin immunoaffinity chromatography, reducing dimeric inhibin A and B by >90% and abolishing inhibin bioactivity. These preparations were investigated using cultures of rat pituitary cells. GnRH-induced LH and FSH secretion in vitro was reduced by ovine corpus luteum extract and human follicular fluid by 47+/-5% and 42+/-5% of control LH and by 37+/-5% and 50+/-10% of control FSH, respectively (P<0.001). Extracts prepared from corpora lutea and placentae that were collected on days 50, 90 and 120 of pregnancy (five ewes per stage of pregnancy) showed increased GnRH-induced LH-suppressing bioactivity, particularly in the case of the placental extracts, with a threefold increase in activity. When partially purified by pseudochromatofocusing, GnRH-induced LH-suppressing bioactivity in extracts of ovine corpora lutea was identified at pH 5.40 and 5.77. Although these values are similar to published gonadotrophin surge-attenuating factor (GnSAF) bioactivity pI values, a GnSAF-blocking antiserum had no consistent effect on ovine corpus luteum extract GnRH-induced LH-suppressing bioactivity. It was concluded that the ovine corpus luteum of pregnancy contains a non-steroidal, non-inhibin factor, probably not GnSAF, that has the ability to reduce pituitary responsiveness to GnRH in vitro.

Demonstration of a non-steroidal, non-inhibin factor in the ovine corpus luteum of pregnancy that reduces pituitary responsiveness to GnRH-induced LH secretion in vitro

The decline in pulsatile LH secretion and pituitary responsiveness to GnRH as pregnancy advances may be due to non-steroidal factors secreted by the ovine corpus luteum of pregnancy. Corpora lutea from ten ewes on days 70-80 of gestation were homogenized, charcoal-treated and, together with charcoal-treated follicular fluid from superovulated women, were subjected to inhibin immunoaffinity chromatography, reducing dimeric inhibin A and B by >90% and abolishing inhibin bioactivity. These preparations were investigated using cultures of rat pituitary cells. GnRH-induced LH and FSH secretion in vitro was reduced by ovine corpus luteum extract and human follicular fluid by 47+/-5% and 42+/-5% of control LH and by 37+/-5% and 50+/-10% of control FSH, respectively (P<0.001). Extracts prepared from corpora lutea and placentae that were collected on days 50, 90 and 120 of pregnancy (five ewes per stage of pregnancy) showed increased GnRH-induced LH-suppressing bioactivity, particularly in the case of the placental extracts, with a threefold increase in activity. When partially purified by pseudochromatofocusing, GnRH-induced LH-suppressing bioactivity in extracts of ovine corpora lutea was identified at pH 5.40 and 5.77. Although these values are similar to published gonadotrophin surge-attenuating factor (GnSAF) bioactivity pI values, a GnSAF-blocking antiserum had no consistent effect on ovine corpus luteum extract GnRH-induced LH-suppressing bioactivity. It was concluded that the ovine corpus luteum of pregnancy contains a non-steroidal, non-inhibin factor, probably not GnSAF, that has the ability to reduce pituitary responsiveness to GnRH in vitro.

The vascular endothelial growth factor/fms-like tyrosine kinase system in human ovary during the menstrual cycle and early pregnancy.

In human ovaries, angiogenesis is known to be associated with the development of follicles and the formation of the corpus luteum (CL). A complex vascular network is formed within the thecal cell layer during follicular growth, and rapid neovascularization occurs toward the granulosa cell layer after ovulation. Vascular endothelial growth factor (VEGF) is a multifunctional cytokine, stimulating endothelial cell growth and enhancing microvascular permeability. A specific receptor for VEGF, fms-like tyrosine kinase (Flt-1), is expressed in vascular endothelial cells that mediates the action of VEGF. We examined the localization and expression of VEGF and Flt-1, using an immunohistochemical technique and RT-PCR analysis, in human follicles and corpora lutea during the normal menstrual cycle and early pregnancy. We measured concentrations of VEGF in extracts of human CL using an enzyme-linked immunosorbent assay during the luteal phase and early pregnancy. Immunostaining for VEGF was observed in granulosa cells from small antral follicles to preovulatory follicles. The staining was detected in thecal cells from medium-sized to preovulatory follicles. The intensity of the staining was gradually increased as a follicle grew. Flt-1 was localized in granulosa and thecal cells of preovulatory follicles as well as in endothelial cells. In the human CL, the intense staining for VEGF was observed in granulosa and thecal lutein cells, especially in the midluteal phase. The immunostaining for Flt-1 was faint in endothelial cells in the CL, whereas it was distinct in granulosa and thecal lutein cells. The concentrations of VEGF in lutein extracts were high in the early and midluteal phases and tended to decrease toward the late luteal phase. During early pregnancy, a measurable amount of VEGF was detected. RT-PCR analysis demonstrated that messenger ribonucleic acids encoding VEGF121, VEGF165, and Flt-1 were expressed in the CL. These results suggest that VEGF might have an autocrine role in the ovulatory process and luteal function as well as a paracrine role in angiogenesis.

The physiology of ovarian oxytocin

The notion of an oxytocic principle residing within the ovary is not new. In as early as 1910, Ott and Scott showed that an extract of bovine corpus luteum could induce milk letdown and uterine contraction. However, it took a further 70 years before the identification of this principle with the nonapeptide hormone oxytocin (OT) was made at the peptide and mRNA levels. This was followed by the identification of the peptide in ovarian tissues and ovarian venous blood from a wide variety of species, including humans, monkeys, pigs and ruminants (reviewed in 7, 8). For the majority of non-ruminant species the levels of expression of the peptide and its specific mRNA are relatively low, implying that whatever function the ovarian hormone has in these species, it is most likely to be at the local, paracrine level. Ruminants are an exception. Cows and sheep both produce very high levels of OT and OT-mRNA – the latter attaining concentrations of approximately 1% of all transcripts – within the corpus luteum of the early oestrous cycle. In ruminants, evolution has culminated in a systemic link between ovarian OT production and OT receptors in the endometrium of the uterus, inducing there the production of prostaglandin-F2∞ (PGF2∞) which completes a positive feedback loop to the ovary by stimulating further OT release (reviewed in 10). It is important to note, however, that natural selection can only act on a preexisting system. In this case, it has developed a systemic endocrine pathway in ruminants from a local ovarian OT system present probably in all mammals. There is even evidence for OT-related peptides, such as mesotocin and vasotocin, within the ovaries of marsupials and chicken, though their function is not known.

Luteinization in primates is accompanied by loss of a 43-kilodalton adenosine 3',5'-monophosphate response element-binding protein isoform.

Although granulosa cell differentiation and corpus luteum function are both regulated by cAMP, there are development-dependent differences, particularly at the level of gene expression and cell proliferation, between the responses of follicular granulosa cells and luteal cells to trophic hormone stimulation. In this study, we sought to determine whether these differences could be due to changes in the cellular expression of cAMP response element (CRE)-binding protein (CREB). Immunocytochemical analysis of macaque ovaries revealed a development-related alteration in the subcellular distribution of CREB-immunoreactive material. Immunoreactive CREB was present in nuclei of follicular granulosa cells from maturing follicles, whereas after ovulation and luteinization, no CREB-immunoreactive proteins were visualized in luteal cell nuclei. Anti-CREB immunoblotting of granulosa cell extracts from macaque preovulatory follicles as well as extracts of granulosa cells from luteinizing human follicles revealed a 43-kilodalton (kDa) protein, a size typical of native CREB. In contrast, whole cell extracts of monkey corpora lutea collected during the early, mid-, and late luteal phases completely lacked a 43-kDa CREB signal. The absence of 43-kDa CREB isoforms in corpora lutea was confirmed using three different antisera directed against different regions of CREB. Using a human collagenase gene CRE to probe Southwestern blots, a 43-kDa CREB was observed in follicular cell extracts, whereas no CRE-binding activity was found in corpora lutea extracts using this probe. We also sought to determine whether the loss of expression of the 43-kDa CREB isoform may be functionally correlated with the cessation of cellular proliferation that accompanies luteinization. Expression of proliferating cell nuclear antigen (PCNA), an obligatory component of DNA polymerase delta, is essential for proliferation and has been shown by others to be CRE dependent. Immunoblotting of follicle cell and luteal cell extracts with an anti-PCNA monoclonal antibody revealed PCNA expression in granulosa cells and no detectable PCNA expression in corpora lutea. These findings indicate that as follicular granulosa cells progress from the proliferative state to terminally differentiated luteal cells, there is a cessation of expression of a 43-kDa member of the CREB family of transcription factors, and there may be an association between the loss of CREB isoforms and cessation of PCNA expression.

Growth stimulation of ovarian and extraovarian mesothelial cells by corpus luteum extract

Ovarian (OM) and extraovarian (EM) mesothelia represent a common source of gynecologic malignancies with yet unclear pathogenesis. Ovulation triggers a finite wave of DNA synthesis and morphogenesis only in native OM cells, probably through the activation of intraovarian growth factors. To evaluate their growth response to such factors, OM and EM cells were obtained from estrous New Zealand white rabbits by enzymatic dispersion and unit gravity sedimentation. Cell cultures were maintained in serumless, fibronectin-rich, HL-1 medium without or with rabbit corpora lutea tissue extracts (CLE). The growth effects of CLE were evaluated by measuring percent changes in cell number relative to controls (CCN), cell population doublings (CPD), cell population doubling time in hours (CPDT). After 7.5 days, CLE enhance (P < 0.001) the growth of both OM and EM cells, which exhibited, respectively, a CCN of 214 and 257%; a CPD of 2.89 and 2.87; and a CPDT of 54.39 and 59.49. CLE-treated OM and EM cells were smaller, formed more cohesive monolayers, and exhibited more frequent and diffuse microvilli than control cells. These data show a similar in vitro response of OM and EM cells to luteal growth factors, suggesting that the lack of postovulatory morphogenesis in native extraovarian mesothelia is due to the spatially restricted activity of intraovarian growth factors.

Apparent alpha-inhibin subunit immunoactivity in porcine and ovine luteal extracts is due to interference by cytosolic proteases in the assay.

Immunoreactive alpha-inhibin (ir-inhibin) was measured in luteal homogenates and subcellular fractions of ovine and porcine corpora lutea (CL) and in pig granulosa cells (GCs), using a sensitive radioimmunoassay specific for the 1-26 amino acid sequence of the N-terminus of the alpha chain of porcine inhibin (p1-26 alpha-inhibin). Inclusion of N-ethylmaleimide (N-EM) and/or EDTA in the immunoassay had no effect on the measurement of p1-26 alpha-inhibin peptide standards, on ir-inhibin levels in ovine follicular fluid and serum, or on ir-inhibin in subcellular fractions of pig GC. Fractionation of porcine GC homogenates on sucrose gradients demonstrated a major particular peak of ir-inhibin (buoyant density, 1.15-1.21 g/cm3) with variable activity in the cytosol. The particulate ir-inhibin peak was released into the cytosol by pretreatment of GC homogenates with the saponin, digitonin, prior to fractionation. Porcine GC extracts contained a protein (M(r) 45,000) which immunoblotted against p1-26 alpha-inhibin antibody. In the absence of inhibitors of proteolysis, apparent ir-inhibin activity was very high in extracts of sheep and pig CL. However, inclusion of N-EM or EDTA in the radioimmunoassay significantly reduced ir-inhibin levels in porcine and ovine CL extracts in a dose-dependent manner. Measurements of peptide tracer integrity indicated that porcine luteal cytosol degraded 125I-labelled p1-26 alpha-inhibin peptide. Subcellular fractionation studies demonstrated high levels of apparent ir-inhibin in luteal cytosol fractions, with only minor activity peaks associated with particulate fractions; however, this material was not releasable by digitonin. Immunoblotting of detergent extracts of porcine luteal particulate fractions failed to demonstrate alpha-inhibin material, and immunocytochemical localization studies of alpha-inhibin in porcine and ovine luteal sections were negative. Our results are consistent with the intracellular packaging/storage of a form of alpha-inhibin (M(r) similar to that of alpha-inhibin subunit precursor) in the porcine granulosa cell. However, luteinization of the porcine follicle was associated with a dramatic fall in ir-inhibin content, and the loss of immunostaining for alpha-inhibin peptides. We conclude that porcine and ovine CL contain little, if any, authentic inhibin. These studies emphasize the importance of excluding proteolytic artefacts when measuring biological peptides in luteal tissue extracts by radioimmunoassay.

Biosynthesis and release of oxytocin by granulosa cells derived from preovulatory bovine follicles: effects of forskolin and insulin-like growth factor-I.

Granulosa cells derived from preovulatory bovine follicles were cultured in the presence of insulin-like growth factor-I (IGF-I, 10-100 ng/ml), forskolin (10 microM), or a combination of the two agents. Forskolin alone was the most potent stimulator of both oxytocin (OT) and progesterone (P4) secretion. The two hormones had different patterns of secretion during the course of incubation. OT production peaked on Day 5 of culture and declined thereafter, whereas P4 rose gradually to a peak between Days 7 and 9. The addition of IGF-I to forskolin did not augment OT release beyond that achieved with forskolin alone, but it did maintain higher levels of OT secretion beyond the Day-5 peak. Two antisera, (antiserum I and antiserum II) directed against OT and its C-terminally extended forms, respectively, were used to identify the OT forms in culture media and granulosa cell and corpus luteum extracts. Fully processed OT was detected only in small amounts (0.43 ng/mg protein) in granulosa cell extracts, whereas the corpus luteum extracts contained 6 ng/mg protein. However, granulosa cells that had been incubated with forskolin contained stores of the OT precursor oxytocin-neurophysin, which is found in young corpora lutea. These data indicate that forskolin (whose action probably mimics gonadotropin action) is an effective stimulator of OT biosynthesis and release in cultured bovine granulosa cells.

COOH-terminally-extended processing forms of oxytocin in human ovary

Human granulosa cells synthesize and secrete the oxytocin hormone. We have already shown that oxytocin-Gly, the last post-translational maturation intermediate of pro-hormone, is largely secreted by cultured granulosa cells deprived of ascorbate (Plevrakis et al. (1990) J. Endocrinol. 124, R5–R8). Using a combination of high performance liquid chromatography and radioimmunoassay, the oxytocin-like material present in human granulosa cell extracts, in follicular fluid, in cultured granulosa cell supernatants and in corpora lutea extracts was identified. We have demonstrated the presence of oxytocin-Gly, oxytocin-Gly-Lys and oxytocin-Gly-Lys-Arg, the same post-translational maturation intermediates as those we identified in bovine corpus luteum secretory granules. Thus we conclude that post-translational maturation of pro-oxytocin/neurophysin in human ovary proceeds by the same proteolytic events as those we described in bovine post-pituitary gland and corpus luteum.

Measurement of oxytocin concentrations in plasma and ovarian extracts during the oestrous cycle of mares

Jugular venous blood samples were collected throughout a complete oestrous cycle from 9 mares for measurement of progesterone and oxytocin by radioimmunoassay. Mean oxytocin concentrations remained at approximately 1 pg/ml throughout, with no evidence of cyclic variation in the release pattern. Extracts of corpus luteum and follicles obtained from a further 33 mares at different stages of the cycle all contained oxytocin concentrations of less than 10 pg/g wet weight of tissue. We conclude that the ovaries are not a source of circulating oxytocin during the oestrous cycle in this species. The plasma oxytocin concentrations reported here are substantially lower than those found by other groups.

Atrial natriuretic peptide in bovine corpus luteum.

Atrial natriuretic peptide (ANP) has been demonstrated to exert endocrine functions, including the modulation of steroid synthesis. This prompted investigations to search for ANP receptors in the corpus luteum, a tissue that produces progesterone. The studies revealed a single binding site for [125I] ANP with similar characteristics (Kd, 122 pM; maximum binding, 18 fmol/mg protein) in all four stages of corpus luteum development. These receptors were demonstrated to stimulate cGMP production upon activation with synthetic ANP. Maximal cGMP synthesis was observed at 10(-7) M ANP, 5 min after activation of receptors. An acidic extract of corpus luteum contained immunoreactive ANP (approximately 220 fmol/g tissue), as indicated by gel chromatography, HPLC, and identification by means of a highly specific ANP antibody. The data do not permit definition of a specific endocrine role of ANP in the corpus luteum.

Mesotocin and luteal function in macropodid marsupials.

Pituitary glands and corpora lutea collected at various stages of the reproductive cycle of the tammar wallaby (Macropus eugenii), were extracted and fractionated by high-performance liquid chromatography, and specific radioimmunoassays were used to measure mesotocin ([Ile8]-oxytocin) and oxytocin. Mesotocin, but not oxytocin, was identified in extracts of pituitary; the mean concentration of mesotocin in this tissue was 0.75 nmol/g wet weight. Neither mesotocin nor oxytocin was detected in extracts of corpus luteum. In female Bennett's wallabies passively immunized against mesotocin during seasonal reproductive quiescence, there was no significant effect on peripheral progesterone concentrations and there were no births, matings or changes in vaginal smears in the 2 months following treatment. Thus mesotocin is unlikely to act as a systemic luteostatic agent during seasonal quiescence.

Inhibitory effect of oxytocin and vasopressin on steroid release by cultured porcine luteal cells.

Previously, we have demonstrated the presence of substances reacting like arginine vasopressin (AVP) and oxytocin (OXT) in acid extracts of corpora lutea (CL) of pigs by RIA. The present study examined purified extracts of CL by using HPLC. The results of these experiments show that CL of nonpregnant sows contain AVP and OXT. Little is known about possible auto- and paracrine effects of AVP and OXT in the ovary. Therefore, we investigated the influence of AVP and OXT on progesterone, estradiol, and androstenedione secretion in porcine luteal cell cultures from nonpregnant sows. Progesterone and androstenedione secretion increased significantly (P less than 0.05) in the presence of ovine LH (oLH), whereas no change in basal estradiol levels could be observed under the same conditions. When AVP or OXT was added to the culture system a dose-dependent inhibition of basal as well as oLH-stimulated progesterone secretion was measured. Under basal conditions, a dose of 1 pg AVP/ml decreased progesterone secretion significantly (P less than 0.05), but to reach the same effect in the presence of OXT a dose of 100 ng/ml was necessary. In the presence of oLH the addition of as little as 0.01 pg AVP/ml inhibited progesterone secretion significantly (P less than 0.05). On the other hand, 10 ng OXT/ml or higher doses were needed to decrease oLH-stimulated progesterone release. In the presence of specific peptide antagonists the inhibitory effect on progesterone release was abolished. These results suggest that AVP and OXT effects are mediated through specific receptors. OXT and AVP also inhibited androstenedione secretion, but had no effect on estradiol secretion. Calculation of the ED50 data from dose-response curves of both peptides show that AVP is about 10(4)-fold more active than OXT in inhibiting in vitro progesterone and androstenedione secretion. This suggests that AVP as well as OXT may play an important role in the regulation of ovarian function.

Historical retrospective. Lipamin (Agomensin) and luteolipoid (Sistomensin). Animal experiments and preliminary clinical experiences

In 1914 "Lipamin" and "Luteolipoid", two extracts of corpus luteum, were developed at the University Gynaecological Hospital in Erlangen, Bavaria. The process of their preparation, protected by patents, was exploited soon after that by Ciba, Basel, who named their products Agomensin and Sistomensin. The history of their development, their effects in animal experiments, as well as their early therapeutical application to women are reported. Special reference is made to the patent specifications and to the lectures held at meetings of the Bavarian Society of Gynaecology and Obstetrics.

Effects of oxytocin-antagonist injections on luteal regression in the goat.

Intra-arterial administration of 0.25 ml physiological saline to the non-pregnant goat between days 12 and 20 of the oestrous cycle did not affect luteal regression, which was characterized by decreasing peripheral plasma progesterone concentration, beginning on day 13 of the oestrous cycle, and an increase in the plasma concentration of 13, 14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) as oestrus approached on about day 20. Intra-arterial administration of oxytocin antagonist (OA) in saline at a dose of 0.2 microgram kg-1 body weight to goats between days 12 and 20 of the cycle significantly (P less than 0.001) delayed luteal regression beyond day 20 (to day 26). Injection of OA maintained plasma progesterone secretion at 4-5 ng ml-1 till day 23 of the cycle and suppressed the increase in PGFM concentration. Corpus luteum extract (100 microliters) of OA-treated animals released a significant (P less than 0.001) amount of PGF2 alpha from rat uterus in vitro as did authentic oxytocin. This oxytocic material failed to release PGF2 alpha during luteolysis in the goat, suggesting that oxytocin receptors for PGF2 alpha release may be occupied by OA. It is concluded that oxytocin-receptor interaction in the uterus may be the stimulus for PGF2 alpha release which triggers luteal regression in the goat.

A renin-like enzyme in luteal tissue

The aim of this study was to identify immunologically and biologically a renin-like enzyme (RLE) in rat corpora lutea (CL). The biological activity of partially purified extracts of CL was tested in vivo by injection into anesthetized pentolinium-treated rats, obtaining a pressor response similar to renal renin. The enzyme activity in vitro was inhibited to about 50% by pretreatment with a specific antibody against renal renin. When the extracts were incubated with angiotensinogen, the product was inhibited mainly by angiotensin I antibody. The fact that there was no change in RLE content in 24 or 48 h nephrectomized rats, suggested the idea of a local production rather than an active blood renin sequestration.