Ovarian Substance Research Articles

To the treatment of ovarian substance of painful phenomena of natural and premature termination of the month

(Journal. Medicine. Chemistry and Pharmacy December 1896. p. 747).

Gonadotropin Surge-Attenuating Factor: A Nonsteroidal Ovarian Hormone Controlling GnRH-Induced LH Secretion in the Normal Menstrual Cycle.

Gonadotropin surge-attenuating factor (GnSAF) is a nonsteroidal ovarian substance, which attenuates the endogenous LH surge in superovulated women. Different molecular sequences have been found, but only one of them has shown substantial homology to a known substance of the human genome. A molecular mass of 12.5kDa showing identity to the carboxyl-terminal fragment of human serum albumin and expressing GnSAF bioactivity in vitro has been identified. It has been suggested that in the normal menstrual cycle the in vivo bioactivity of GnSAF increases under the influence of the intercycle rise of FSH. GnSAF is considered the "missing link" between the ovaries and the hypothalamo-pituitary system, maintaining the pituitary in a state of low responsiveness to GnRH in the early- to midfollicular phase of the cycle. A marked decline in GnSAF bioactivity in the late follicular phase facilitates the onset and the full expression of the midcycle LH surge.

Long term estradiol-17β administration changes population of the dorsal root ganglia neurons innervating the ovary in the sexually mature gilts

The influence of estradiol-17β (E₂) overdose on the number and distribution of neurons in the dorsal root ganglia (DRGs) supplying the ovary of adult pigs was investigated. The numbers of ovarian substance P (SP)-, calcitonin gene-related peptide (CGRP)-, galanin (GAL)-, pituitary adenylate cyclase-activating polypeptide (PACAP)-, neuronal isoform of nitric oxide synthase (nNOS)- and estrogen receptors (ERs)-immunoreactive perikarya were also determined. On day 3 of the estrous cycle, the ovaries of both the control and experimental gilts were injected with retrograde tracer Fast Blue. From day 4 of the estrous cycle to the expected day 20 of the second studied cycle, the experimental gilts were injected with E₂, while the control gilts received oil. The DRGs Th16-L5 were then collected and processed for double-labelling immunofluorescence. Injections of E₂ increased the E₂ level in the peripheral blood ∼4-5-fold and reduced the following in the DRGs: the total number of Fast Blue-positive perikarya, the number of large perikarya, the population of perikarya in the L2 and L3 ganglia, the numbers of SP- and/or CGRP-, PACAP-, nNOS-immunoreactive perikarya and the number of large perikarya expressing ERs subtype α and β. These results show that long-term E₂ treatment of adult gilts affects both the spatial and neurochemical organization pattern of ovary sensory innervation. Our findings suggest that elevated E₂ levels occurring during pathological states may regulate the transmission of sensory modalities from the ovary to the spinal cord.

FSH modulatory effect on human granulosa cells: a gene–protein candidate for gonadotrophin surge-attenuating factor

Previous evidence indicates a homology of gonadotrophin surge-attenuating factor (GnSAF) to the carboxyl terminal of human serum albumin (HSA) and the ability of human granulosa cells to produce mRNA transcripts corresponding to this fragment, but the underlying mechanism is still unknown. This study investigated the role of FSH in vitro in the expression of the carboxyl terminal of HSA by human luteinized granulosa cells. Cells were cultured on poly- l-lysine-coated microscope slides in the absence or presence of 10 ng/ml FSH, followed by in-situ hybridization and immunocytochemistry. In the presence of FSH, mRNA transcripts corresponding to the carboxyl terminal of the HSA gene and corresponding protein could be detected in comparable intensity to that seen by hepatic HepG2 cells (positive control). Significantly lower expression was detected in granulosa cells cultured without FSH addition ( P < 0.01), but no expression was detected in HeLa cells. These results demonstrate for the first time that FSH stimulates the expression of the carboxyl terminal fragment of the HSA gene and corresponding protein in human luteinized granulosa cells. Therefore, the carboxyl terminal of HSA has a functional role in the ovary and this further supports the notion that this HSA fragment is a GnSAF-bioactive entity. Gonadotrophin surge-attenuating factor (GnSAF) is a nonsteroidal ovarian substance which attenuates the endogenous LH surge in superovulated women. In-vivo and in-vitro evidence has shown that GnSAF is produced under the stimulation of FSH and may play a physiological role during the normal menstrual cycle. We have previously demonstrated that GnSAF has identity to the carboxyl terminal fragment of human serum albumin (HSA). In this study, we investigated the role of FSH on the expression of the carboxyl terminal fragment of HSA by human luteinizing granulosa cells in vitro. It is demonstrated for the first time that FSH stimulates the expression of this fragment of HSA gene and the corresponding protein in human luteinizing granulosa cells. This finding supports further the notion that this fragment of HSA is the GnSAF bioactive entity.

From Menarche to Regular Menstruation

Marked changes in hormone secretion occur from childhood to adulthood. Prior to puberty gonadotropin-releasing hormone (GnRH) secretion is markedly suppressed. At the onset of puberty, the hypothalamic gonadostat is derepressed and the amplitude of GnRH pulses increases. Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels increase gradually during puberty stimulating follicle maturation and estrogen production in the ovaries. Only the negative feedback mechanism is powerful before puberty, while the positive feedback mechanism becomes active for the first time in late puberty. As a result, normal cyclicity is usually established at that time. During normal menstrual cycle, steroidal and nonsteroidal hormones mediate the effect of the ovaries on the hypothalamic-pituitary system. Estradiol and progesterone are important regulators of FSH and LH secretion, while inhibins play a role in the control of FSH secretion. Gonadotropin surge attenuating factor (GnSAF) is a nonsteroidal ovarian substance that controls the amplitude of the midcycle LH surge by antagonizing the sensitizing effect of estradiol on the pituitary.

Ovarian feedback, mechanism of action and possible clinical implications

The secretion of gonadotrophins from the pituitary in women is under ovarian control via negative and positive feedback mechanisms. Steroidal and non-steroidal substances mediate the ovarian effects on the hypothalamic-pituitary system. During the follicular phase of the cycle, estradiol (E(2)) plays a key role, while circulating progesterone (at low concentrations) and inhibin B contribute to the control of LH and FSH secretion respectively. During the luteal phase, both E(2) and progesterone regulate secretion of the two gonadotrophins, while inhibin A plays a role in FSH secretion. The intercycle rise of FSH is related to changes in the levels of the steroidal and non-steroidal substances during the luteal-follicular transition. In terms of the positive feedback mechanism, E(2) is the main component sensitizing the pituitary to GnRH. Activity of a non-steroidal ovarian substance, named gonadotrophin surge-attenuating factor (GnSAF), has been detected after ovarian stimulation. It is hypothesized that GnSAF, by antagonizing the sensitizing effect of E(2) on the pituitary, regulates the amplitude of the endogenous LH surge at midcycle. Disturbances in the feedback mechanisms can occur in various abnormal conditions or after treatment with pharmaceutical compounds that interfere with the production or the action of endogenous hormones.

Modulatory Effect of the Ovary on LH Secretion

Ovarian steroids are important regulators of gonadotropin secretion during the normal menstrual cycle. Estradiol is the predominant factor in the follicular phase, while progesterone plays an important role in the luteal phase. Gonadotropin surge attenuating factor (GnSAF) is a nonsteroidal ovarian substance that attenuates the endogenous LH surge in superovulated women. Attempts to purify GnSAF from various sources have provided several sequences. In human follicular fluid, GnSAF is shown to be the carboxyl terminal fragment of human serum albumin (HSA) and a molecular mass of 12.5 kDa. Preliminary data have shown the expression of the carboxyl terminal fragment of HSA in human granulosa cells. In vivo studies have provided evidence that GnSAF may participate in the control of LH response to GnRH in the context of a mechanism that facilitates the full expression of the midcycle LH surge. Further research is expected to clarify the role of GnSAF in women's physiology.

Hybridization barriers between wheat and rye: In vitro pollen assays and electrophoretic survey

Five- to seven-day-old ovaries from Triticum aestivum vars Hope (non-crossable), Chinese Spring (CS, crossable) and the chromosomal substitution line Chinese Spring/Hope 5B (CS/H5B) were used to test the hypothesis that an ovarian substance inhibits rye (Secale cereale var Wrens) pollen tube growth. In vitro assays on a semi-solid medium demonstrated that a soluble, dialyzed lysate from Hope or CS/H5B ovaries inhibited rye pollen tube elongation significantly more than a similar lysate from CS ovaries (26%, 13% and 5% respectively compared to controls). When only the 20 longest tubes from each sample were included in the analysis the relationship was maintained (33%, 22% and 15% for Hope, CS/H5B and CS respectively). Heating the Hope lysate at 100° C for 10 min reduced the inhibition from 26% to 14% for all pollen tubes measured (n = 51 per sample) and from 33% to 20% when only the 20 longest pollen tubes per sample were analyzed. Isoelectric focusing followed by sodium dodecyl sulfate polyacrylamide electrophoresis (IEF/SDS-PAGE) and non-equilibrium pH gradient electrophoresis (NEPHGE) revealed several differences in the polypeptide profiles of soluble lysates from vars Hope, CS and CS/H5B. While two striking differences were observed, a 50 kilodalton (kD) polypeptide with an isoelectric point (pI) of approximately 8.5 and 100 kD polypeptide (pI∼4) from CS lysates which were considerably reduced in lysates of Hope and CS/H5B, none were obviously associated with the Hope 5B chromosome and therefore cross-incompatibility.

More Yellow Ovaries

To the Editor.— A case of unilateral yellow pigmentation of the ovary was reported recently. 1 We wish to add two cases in which the total ovarian substance was pigmented; neither case was associated with symptoms or pathological findings. Report of Cases.— Case 1.— A 29-year-old woman presented with primary infertility of 6 years' duration. Her cycle was regular, with occasional midcycle ovulatory bleeding. She had been examined outside the United Kingdom, with a dilatation and curettage reported as normal and a hysterosalpingogram that apparently showed bilateral tubal obstruction. She was admitted for laparoscopy, dilatation and curettage, and a dye test, which showed a uterus enlarged to about 8 weeks' size by multiple small fibroids. The tubes looked normal, but there was no spill of dye. The ovaries were of normal dimensions, the left containing a corpus luteum, but both were bright orange-yellow. The dilatation and curettage produced

Embryonic loss in mares: Nature of loss after experimental induction by ovariectomy or prostaglandin F 2α

Twenty-one pregnant pony mares were assigned to one of the following groups: 1) controls, 2) ovariectomy at Day 12, 3) ovariectomy at Day 12 plus daily progesterone treatment on Days 12 to 40, 4) PGF 2α on Day 12, 5) PGF 2α on Day 21, and 6) PGF 2α on Day 30. Based on daily examinations by ultrasound, the embryonic vesicle was maintained to Day 40 in all control mares and in mares that were ovariectomized on Day 12 and given progesterone. The embryonic vesicle was lost in all mares of the other four groups. Administration of progesterone prevented the embryonic loss associated with ovariectomy at Day 12, indicating that progesterone may be the only ovarian substance required for survival of the early embryo. The mean number of days to embryonic loss was greater for mares treated with PGF 2α on Day 12 (6.8 days) than for mares ovariectomized on Day 12 (3.0 days). In the PGF 2α-treated group, the vesicles did not become fixed at the expected time (Day 15), and mobility continued until the day of loss. In the mares treated with PGF 2α on Day 21 and in one of the mares treated on Day 30, the vesicle was lost within one to three days without prior indication. Loss may have occurred by expulsion through the cervix, since the cervix was patent on the day of loss in these mares and in the mares ovariectomized or treated with PGF 2α on Day 12. In the remaining mares treated on Day 30, the intact embryonic vesicle was dislodged on Day 31 or 32. The dislodged vesicle was mobile within the uterus and was frequently found in the uterine body. The fluid volume of the dislodged vesicle gradually decreased, and the fluid was no longer detected by Day 38 to 42. Some of the placental fluids may have been eliminated by resorption since the cervix remained closed while the fluid volume decreased.

Low molecular weight substance from rat ovary induces steroidogenesis in cultured granulosa cells

Ovaries from immature intact rats contain an apparently low molecular weight substance which mimics the action of follitropin (FSH) on ovarian granulosa cells in culture. Similar to FSH action, the ovarian substance (OS) induced cell-shape changes followed by intensive progestin production. Like FSH action, OS-induced steroidogenesis reversibly ceased upon washing the factor from the cultured cells, and could be blocked in the presence of cycloheximide or α-amanitin. Although OS stimulated aromatase activity in granulosa cells, it failed to elicit LH responsiveness in the cultured cells. Androstenedione synergistically augmented OS-induced progestin production and aromatase activity. OS itself synergistically augmented FSH-induced progestin but did not have any effect on FSH-induced aromatase activity. In contrast to FSH action which is mediated via cAMP formation, OS doses which evoked extensive synthesis of progestin products failed to stimulate significant increases in intracellular cAMP accumulation. These results suggest the existence of a putative intraovarian hormone-like substance which can mimic some effects of the gonadotropins on the follicular granulosa cell differentiation and may facilitate FSH action at yet unknown stages of the follicular development.

Ovarian substance induces steroid production in cultured granulosa cells.

The rat ovary produces an apparently low molecular weight substance that mimics the action of follitropin (FSH) on ovarian granulosa cells in culture. Similar to FSH action, the ovarian substance (OS) induces temporal cell rounding and, later on, intensive progestin production. However, unlike FSH, OS does not induce accumulation of cyclic AMP (cAMP) in the granulosa cells. The ovarian factor cannot be cAMP as its action is not abolished by phosphodiesterase (PDE) treatment. Neither is it a possible PDE inhibitor, as it does not augment cAMP accumulation in granulosa cells or Friend erythroleukemic cells induced by FSH or PGE1, respectively. The factor is still active after heating for 20 min at 90 degrees C but is rapidly inactivated by alkali treatment. In addition, treatment with various proteases did not abolish the steriodogenic activity. These findings suggest a possible novel intraovarian regulator of the granulosa cell function.

Modulation of fsh-controlled steroidogenesis in rat granulosa cells: Direct in-vitro effects of LHRH and ICI-118630

Direct inhibitory effects of LHRH and an LHRH agonist (ICI-1186 30) on FSH-controlled steroidogenic processes in ovarian granulosa cells were characterized in vitro. Over a 2-day culture period in the presence of testosterone (10 −7 M), FSH (3–3000 ng/ml) caused dose-dependent increases in the aromatase activity of granulosa cells isolated from oestrogen-pretreated immature rats. Progestogen biosynthesis was stimulated in a similar manner. The presence of LHRH (10 −9–10 −7 M) in the culture medium inhibited these responses by right-shifting the dose-response curves. Thus the net effect was one of reduced sensitivity to FSH. ICI-118630 was approximately 10 times more effective than LHRH as an inhibitor of aromatase induction and progestogen biosynthesis in response to FSH. Over a 1-h incubation at concentrations up to 10 −7 M, neither decapeptide had a consistent inhibitory effect on FSH-stimulated granulosa cell cAMP formation either in the presence or absence of 1-methyl-3-isobutyl-xanthine (MIX); but during the 2-day culture, ICI-118630 and occasionally LHRH significantly inhibited aromatase induction by cholera toxin and 2 different cAMP analogues. Over the same range of concentrations, each peptide progressively inhibited the stimulatory effect of MIX on FSH-induced aromatase activity and progestogen biosynthesis. Thus LHRH/ICI-118630 can directly modulate FSH-controlled granulosa cell steroidogenesis in vitro via effects on one or more biochemical loci distal to the FSH-receptor coupled adenylate cyclase system. These experiments have implications for the role of a putative LHRH-like ovarian substance(s) in the local co-ordination of follicular development and function.

The Gonads of the South American Dolphins, Inia geoffrensis, Pontoporia blainvillei, and Sotalia fluviatilis

The appearances of the gonads are described in males and females of 18 Inia geoffrensis, 11 Pontoporia blainvillei, and eight Sotalia fluviatilis from South America. Males of I. geoffrensis become sexually active at a length of about 228 centimeters, females at 175 to 180 centimeters. Length at birth is 76 to 80 centimeters; parturition occurs from about July to September in the upper Amazon. Males of P. blainvillei are still sexually immature at a length of 128.5 centimeters, females become sexually active at a length of 137 centimeters. Off Uruguay, pregnant females have fetuses 6 centimeters in length in February and 61 centimeters in October. Males of S. fluviatilis are sexually active at a length of 148 centimeters, females at 140 centimeters. Gonad weights and details of corpora lutea and albicantia are given. Corpora albicantia appear to persist as in other cetaceans. The ovaries of I. geoffrensis are relatively bulky with the corpora enclosed in the ovarian substance and not pedunculated as in P. blainvillei and S. fluviatilis in which the right ovary is poorly developed.

A histological study of non-follicular cysts in the ovulation fossa region of the equine ovary.

AbstractForty‐two unselected ovaries from adult mares were examined histologically, and with histochemical methods for mucins. A considerable part of the surface of the ovulation fossa was directly covered by columnar epithelium, with many ciliated cells. This epithelium, which was distributed mainly on the anterior side of the ovulation fossa, closely resembled the contiguous epithelium of the infundibulum of the oviduct, was frequently folded, and gave rise to short clefts projecting into the ovarian substance. The remainder of the ovulation fossa was covered by non‐ciliated, low cuboidal or squamous epithelium, lacking folds or clefts.“Fossa cysts,” up to 6.5 mm in diameter, were observed in the ovarian tissue around the ovulation fossa in 27 (64%) of these ovaries. Both simple and branched, tubular and vesicular forms were present, and all were blind‐ending. Their epithelial lining cells, which varied from simple squamous to columnar in type, were frequently ciliated. Many fossa cysts contained secretions histochemically similar to those of the columnar epithelium of the ovulation fossa and infundibulum. Both sialic acidcontaining and neutral mucins were present. It is suggested that these cysts were probably derived by ingrowth from the columnar epithelium of the ovulation fossa. This epithelium may be of müllerian duct origin.

TREATMENT OF HEMORRHAGE IN NONHEMOPHILIC PATIENTS WITH AN ESTROGENIC SUBSTANCE

The primary investigations of Carrol Birch, 1 of Chicago, in 1931 led to the use of ovarian extract in the treatment of hemophilia. Birch reasoned that since the disease occurs only in the male there is something lacking in the male organism which is present in the transmitting female which prevents the development of clinical manifestations of the disease. Working on this theory, she has treated hemophilia with ovarian extract with excellent results, and the use of this in the hands of many others since that time has proved the value of her work. It was also shown that the addition of ovarian substance lowers the coagulation time of blood from a hemophiliac in vitro. At the suggestion of A. J. Carlson, Birch demonstrated that when the urine from normal men is concentrated and injected in sufficient quantities into castrated female rats an estrus cycle is produced. The urine

Ovarian and Uterine Grafts

Recent experience has convinced the author of the value of ovarian grafting. He gives the histories of a number of women upon whom he made implants of their own ovarian tissue in the course of operations for double oöphorectomy. The subsequent state of these patients is contrasted with that of women after castration without grafting. When the uterus was left in position menstruation was re-established in over 80% of the grafted cases.The author and his co-workers maintain that the vitality of autogenous grafts of ovarian substance is enhanced by hormones produced by the uterine mucosa. He therefore advocates that grafts of uterus be made in conjunction with ovarian implants whenever possible. Experiments carried out on animals have proved the correctness of this opinion.The results of a series of cases of combined ovarian and uterine graftings are recorded, and the technique of uterine mucosa implants is described.

OVARIAN THERAPY

The known facts as to the function of the ovary are sufficient to supply a rational basis for ovarian therapy in certain indications. Especially important in this connection is the study of the phenomena produced by complete removal of ovarian tissue, such as the cessation of menstruation, the frequent appearance of troublesome subjective symptoms similar to those of the natural menopause, and the occurrence of certain metabolic changes. The obvious suggestion derived from such observations is that clinical symptoms thought to be due to ovarian deficiency might be cured or relieved by administering some form of ovarian substance. This fundamental idea has been subjected to some refinement as a result of studies aiming to stress some one or other of the ovarian constituents as the source of the active principle. Based on such investigations, several forms of ovarian therapy have developed, including the administration of whole ovary substance, of corpus

Ueber eingebildete Schwangerschaften.

Dr. Hofstätter has presented a timely discussion of pseudocyesis from the standpoint of our most recent conception of its etiology—that of a disturbance of the endocrines. The author accepts the premises that, in view of the hypertrophy of the anterior lobe of the hypophysis during pregnancy, with certain physical changes that are quite generally thought to be related to an increased secretion of that structure, pseudocyesis is incident to some hypophysial change—with a coincident dysfunction of the ovarian substance. In other words, the association of increased activity of the hypophysis, with some abnormality of function of the ovary, produces the symptom complex of pregnancy without the existence of pregnancy. We are fully in accord with the author in the general assumption that endocrinology explains much in the etiology of many instances of pseudocyesis—the fact that the thyroid and the suprarenals, for example, offer evidences of hypertrophy in true pregnancy would

ACTION OF SOME OVARIAN AND CORPUS LUTEUM EXTRACTS ON THE PUPIL OF THE FROG’S EYE

The authors have been engaged for some time in studying the pharmacological and physiological properties of various glandular extracts. In connection with these studies, systematic observations have been made concerning the effect of such extracts on the pupil of the frog’s eye, inasmuch as it is well known that certain hormones such as epinephrin exert a marked influence on that organ. It is deemed desirable to report in the present communication the findings concerning the action of some ovarian and corpus luteum extracts on the frog’s pupil. The preparations used in this research were the desiccated lutein of the sow prepared by the Hynson, Westcott, and Dunning Company without admixture of any preservatives, and the desiccated ovarian substance of the sow, marketed by Armour and Company. Further experiments were also made with fresh corpus luteum substance of the sow and with the fresh ovary of the same animal. The extracts employed were prepared by treating the glands with cold physiological saline o...