Studies on the purification and properties of teleost prolactin

A protein fraction containing prolactin activity from the pituitary tissue of a teleost fish, $\text{Tilapia mossambica}$, has been purified by a combination of ion-exchange and exclusion chromatographic procedures. The purified $\text{Tilapia}$ prolactin was characterized by disc gel electrophoresis, amino-terminal group identification, and amino acid analysis. Its amino acid composition was found to be similar to ovine prolactin. The purified fish prolactin was found to be 40–50 times more potent than ovine prolactin in the $\text{Tilapia}$ sodium-retaining bioassay. However, it was found to be devoid of $\text{Gillichthys}$ yellow pigment-dispersing activity which was previously thought to be a property of teleost prolactin.     

Stimulation of prostaglandin synthetase activity in rat granulosa cells by gonadotropins in vivo

The effect of $\text{in vivo}$ exposure to gonadotropin on prostaglandin synthetase activity in rat granulosa cells was examined in two experimental settings. The first setting was immature rats treated with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). The second was mature rats on the day of proestrus. In the experiments using immature rats, the administration of hCG (20 I.U.) at noon of the second day after the PMSG (20 I.U.) injection led to large (more than 5 fold) increases in granulosa cell prostaglandin synthetase activity 5 and 10 h later. Follicular fluid PGE levels were also markedly increased at 5 and 10 h after hCG. Similar results were also found in experiments performed with mature proestrus rats. Granulosa cell prostaglandin synthetase activity was elevated at approximately 4 and 8 h after the endogenous LH surge (about 4 p.m. on proestrus), in comparison with the activity at midnight of diestrus, or noon and 4 p.m. on proestrus. In these experiments the changes in prostaglandin synthetase activity (10 fold) also paralleled the increases in follicular fluid PGE concentrations. Thus the exposure to gonadotropin $\text{in vivo}$ produced essentially the same effect as we had reported earlier for isolated granulosa cells incubated with LH $\text{in vitro}$. The stimulation of prostaglandin synthetase activity must therefore be ascribed an important role in the physiological regulation of granulosa cell prostaglandin synthesis by LH.     

The nature of the pituitary gonadotropins and their role in ovulation in a urodele amphibian (Ambystoma tigrinum).

Gonadotropic hormones were fractionated from pituitaries of the urodele amphibian $\text{Ambystoma}$$\text{tigrinum}$ (tiger salamander) by methods previously employed to separate these hormones in other species of tetrapod vertebrates. These procedures yielded two distinct fractions that resembled the FSH and LH from other species in regard to their biological profiles in several nonmammalian bioassays and by their chromatographic behavior. The $\text{Ambystoma}$-FSH was free of LH activity (< 0.1%). The major $\text{Ambystoma}$-LH was highly potent in ovulation assays for LH; it also had a high activity in the $\text{Anolis}$ lizard assay, but it is not clear whether this reflects high intrinsic activity or incomplete separation of FSH.$\text{In vitro}$ studies with these and other (frog, turtle, mammalian) gonadotropins indicate that the induction of ovulation in $\text{Ambystoma}$, as in anurans, is highly specific for LH, independent of the source of the gonadotropins. These data support the view that two separate gonadotropins existed early in tetrapod evolution.     

Stimulation of the estrogen synthesizing system of the immature rat ovary by exogenous and endogenous gonadotropins

Immature rat ovaries increase their secretion of estradiol (E₂) when stimulated by gonadotropins but only after a lag period of several hours. Once established, estrogen secretion can be maintained, or increased, by the continued presence of gonadotropin. A combination of ovine FSH+LH given at 2 hr intervals stimulated the estrogen synthesizing system ($\text{ESS}$) of the ovary and serum E₂ showed a pronounced rise between 16 and 20 hrs after the initial injection. When given every 2 hrs for 5 doses (0–8 hrs) serum E₂ was undetectable. However, it was increased if 20 IU PMS was injected at the time of the last dose of FSH+ LH. Endogenous FSH&LH, increased by hourly injections of LH-releasing hormone for a period of 8 hrs, stimulated the $\text{ESS}$; serum E₂ increased at the expected time when this treatment was followed by an injection of PMS.Anti-PMS antiserum given 12 hrs after PMS, prevented the expected rise in serum E₂ at 24 hrs. However, FSH, LH or a combination of the two given every 2 hrs beginning at the time of the anti-PMS produced an increase in E₂ secretion; the combination was more effective than either hormone alone.These results are consistent with the interpretation that a combined FSH-LH action is responsible for induction of the $\text{ESS}$ in the immature rat ovary. The combination of hormones is also very effective in maintaining estrogen secretion but some function appears possible with FSH or LH alone.     

Synthesis and biological activity of [D-Trp6] chicken luteinizing hormone-releasing hormone

An agonist of chicken hypothalamic luteinizing hormone-releasing hormone (cLH-RH). [D-Trp⁶] cLH-RH, was synthesized and tested for luteinizing hormone (LH)-releasing activity using dispersed chicken anterior pituitary cells, as well as for binding to rat anterior pituitary membrane receptors. cLH-RH and mammalian LH-RH (mLH-RH) gave identical dose-response curves in stimulating chicken LH release ($\text{ED}{}_{50}\text{=1.6}$ and $\text{1.8×10}{}^{\mathrm{?9}}\text{M}$ respectively) and similar estimates of potency. The [D-Trp⁶] analogs of cLH-RH and mLH-RH stimulated LH release at lower doses ($\text{ED}{}_{50}\text{=7.0}$ and $\text{～7.0×10}{}^{\mathrm{?11}}\text{M}$ respectively) and were approximately 20-fold more potent. In contrast to the activity in the chicken bioassay, cLH-RH bound to rat anterior pituitary membrane receptors with a much lower affinity than did mLH-RH and had a relative potency of 2%. [D-Trp⁶] cLH-RH was approximately 100-fold more potent than cLH-RH in the rat receptor assay while [D-Trp⁶] mLH-RH was 28-fold more active than mLH-RH. These data demonstrate that substitution of Gly⁶ of LH-RH with D-Trp enhances the LH release from chicken pituitary cells to a similar extent to that observed in mammals, and indicate that the approaches used to produce active LH-RH analogs in mammals are likely to be applicable to birds.     

Regulation of testicular LH receptors by homologous hormone: in vitro studies on receptor occupancy and receptor loss.

A method is described which makes use of 4M MgCl₂ to dissociate the testicular luteinizing hormone-receptor complex without altering either the binding capacity or binding affinity of the receptor. Using this method, it was demonstrated that $\text{in vitro}$ incubation at 4° of decapsulated rat testes with various concentrations of luteinizing hormone or with human chorionic gonadotropin resulted in a reduction in binding capacity. This reduction of binding capacity could not be completely accounted for by occupation of receptors by homologous hormone, suggesting that receptors were lost. Thus negative regulation of LH receptors by LH and hCG was observed. The reduction in LH binding capacity was specific for LH and hCG, dose dependent and time related. FSH, prolactin and growth hormone did not exert the same effect.     

Effect on an antagonistic analog of gonadotropin releasing hormone upon ovarian granulosa cell function.

We have recently demonstrated that gonadotropin releasing hormone (GnRH) acts directly on ovarian granulosa cells to inhibit the follicle stimulating hormone (FSH)-induced increase in granulosa cell steroidogenesis $\text{in}$$\text{vitro}$. A GnRH antagonist, [D-pGlu¹, D-Phe², D-Trp^3,6] GnRH (A), which is known to antagonize GnRH-stimulated gonadotropin release by cultured pituitary cells, was tested in the granulosa cell system. GnRH (10^?8M) inhibited estrogen and progesterone production by FSH-treated granulosa cells $\text{in}$$\text{vitro}$, whereas the antagonist A (10^?6M) did not affect FSH stimulation of steroidogenesis. Antagonist A, when added together with GnRH and FSH, blocked the GnRH inhibition of FSH-induced steroidogenesis. Estrogen and progesterone production by granulosa cells was increased by 50% at a molar ratio (IDR₅₀) of $\text{20}\text{1}\text{and}\text{12}\text{1}$ ([antagonist]/[GnRH]), respectively. At 10^?6M, antagonist A completely prevented the GnRH (10^?8M) inhibition. A similar effect of antagonist A was seen in FSH-induced increase of luteinizing hormone (LH) receptor content. FSH treatment for 2 days $\text{in}$$\text{vitro}$ induced an 8-fold increase in LH receptor content in cultured granulosa cells; concomitant treatment with 10^?8M GnRH completely inhibited the FSH effect. Antagonist A (10^?6M), by itself, had no effect on the FSH action. However, when added together with FSH and GnRH, antagonist A completely abolished the inhibitory effect of GnRH. These results demonstrate that the direct inhibitory effect of GnRH on granulosa cell function can be prevented by a GnRH antagonist and that the GnRH action at the ovarian level may require stringent stereospecific interactions of these peptides with putative GnRH recognition sites.     

Variations in the number of pituitary LHRH receptors correlated with altered responsiveness to LHRH

Isolated pituitary cells from metestrous, ovariectomized (OVX), and ovariectomized-estradiol treated (OVX-EB) rats were employed to study the gonadotropin response to luteinizing hormone-releasing hormone (LHRH) challenge and to quantitate LHRH receptors, using a labeled LHRH analog. Ovariectomy (3–4 weeks post castration) resulted in a reduction of LHRH receptor concentration from 34.4 ± 2.1 in metestrous females to 14.3 ± 0.9 fmoles/10⁶ cells. Concomitantly, the luteinizing hormone (LH) response to a near-maximal dose of LHRH (5 ng/ml) decreased from a 3-fold stimulation in intact females to 1.13-fold stimulation in cells from OVX rats. Replacement therapy with EB (50 ug/rat for 2 days) to OVX rats restored LH response and LHRH binding sites (a 2.5-fold stimulation in LH secretion and 32.0 ± 2.1 fmoles/10⁶ cells, respectively). The LH response to LHRH stimulation was not altered after one day of EB treatment although the number of LHRH binding sites was increased. The changes in the number of LHRH binding sites were not accompanied by any alterations in the affinity of the LHRH analog ($\text{K}{}_{\mathrm{d}}\text{? 0.5 × 10}{}^{\mathrm{?9}}\text{M}$). It is concluded that variations in LHRH receptor number reflect the degree of pituitary sensitivity to LHRH and it may suggest that LHRH and estradiol modulation of gonadotropin release is mediated by these receptors.     

Role of prostaglandin F2α in modulation of LH-stimulated steroidogenesis in vitro in different types of rat and ewe corpora lutea

An inhibitory effect of PGF_2α at a dose of 7 × 10^?7 M on LH stimulated synthesis of progesterone was observed $\text{in vitro}$ after incubation of pseudopregnant rat ovaries for a period of 2 hours. A similar effect was seen with cyclic and gestant ewe corpora lutea at the same dose of PGF_2α. This effect was observed both in the secretion of progesterone and on the amount of progesterone present in the tissue. This inhibitory effect of PGF_2α on LH stimulated progesterone synthesis may explain the modification in the time course for gonadotropin action in luteal tissue at high and low doses.     

Response of superfused goldfish pituitary fragments to mammalian and salmon gonadotropin-releasing hormones

Salmon and mammalian gonadotropin-releasing hormones (sGnRH, mGnRH) were tested for their ability to stimulate $\text{in vitro}$ gonadotropin (GtH) release from superfused goldfish pituitary fragments. A two minute exposure to either peptide was sufficient to stimulate a dose-dependent increase in GtH release which reached maximum levels in 15 minutes and returned to baseline within one hour. Both peptides were approximately equipotent in stimulating GtH release, as was a superactive analog of mGnRH. These results demonstrate that sGnRH is capable of directly stimulating GtH release from teleost pituitary tissue, and that structural differences between the three peptides tested do not result in significant differences in $\text{in vitro}$ bioactivity.     

Coincidence of down-regulation and desensitization in pituitary gonadotrophs stimulated by gonadotropin releasing hormone

The dynamics of gonadotropin releasing hormone (GnRH) induced luteinizing hormone (LH) release was studied $\text{in}$$\text{vitro}$ by superfusion of cultured pituitary cells. Continuous exposure of the cells to GnRH resulted in desensitization of the gonadotroph responsiveness to further stimulation by the hormone. The refractory state was achieved within 4 hr of hormone introduction (10^?7 M) and was accompanied by down-regulation of GnRH receptors (50%) assayed by equilibration with [¹²⁵I]iodo-[D-Ala⁶]des-Gly¹⁰-GnRH N-ethylamide. The data indicate that GnRH can regulate the number of its own receptors, and that desensitization is accompanied by down-regulation.     

Luteinizing hormone (LH) release after single injections of a synthetic LH-releasing hormone (LH-RH) in the ewe at three different reproductive stages and comparison with natural LH release at oestrus

The possibility was investigated of using single i.v. injections of a synthetic luteinizing hormone-releasing hormone (LH-RH) to manipulate the reproductive pattern of the ewe.Single i.v. injections of 150 μg synthetic LH-RH were given on Day 12 of the oestrous cycle, during seasonal anoestrus and on Day 16 $\text{post}$-$\text{partum}$ in ewes which lambed during the breeding season. Blood samples were obtained at 5-, 10- or 15-minute intervals for 1 hour before and for 3 hours after treatment. Plasma LH concentrations were measured using a specific double antibody radioimmunoassay, the development of which is described. Laparotomy was performed on each animal 2–3 days after treatment.The treatment induced LH peaks in all animals and ovulation in the majority. There was no significant difference between the groups in the LH response. The LH release was, however, much less than that found in untreated ewes sampled every 15 minutes for 18 hours during oestrus.     

Localization and in vitro synthesis of prostaglandins in components of rabbit preovulatory Graafian follicles

Graafian follicles obtained 9 hours after the injection of human chorionic gonadotropin (hCG) into mature rabbits were dissected into a follicular fluid component, a granulosa cell-oocyte component, and a residual wall component, (the latter containing mostly theca tissue with a small and variable amount of adhering granulosa cells). The amounts of PGE and PGF were determined for each component. The follicular fluid contained approximately 4–10 times more PGE and PGF than either the granulosa cell-oocyte component or the residual wall component. The latter two components contained approximately equal amounts of these prostaglandins. The $\text{in vitro}$ biosynthesis of PGE and PGF was also studied and it was found that the granulosa cell-oocyte component had about 4 fold the capacity of the residual wall, and that the follicular fluid synthesized no prostaglandins. There was no significant effect of LH on either PGE or PGF synthesis in any of the components.     

Effects of intraventricular injection of gastrin on release of LH,prolactin, TSH and GH in conscious ovariectomized rats

Conscious ovariectomized (OVX) rats bearing a cannula implanted in the 3rd ventricle were injected with 2 μl of 0.9% NaCl containing varying doses of synthetic gastrin and plasma gonadotropin, GH and TSH levels were measured by RIA in jugular blood samples drawn through an indwelling silastic catheter. Control injections of saline iv or into the 3rd ventricle did not modify plasma hormone levels. Intraventricular injection of 1 or 5 μg gastrin produced significant suppression of plasma LH and prolactin (Prl) levels within 5 min of injection. Injection of 1 μg gastrin had no effect on plasma GH, but increasing the dose to 5 μg induced a progressive elevation, which reached peak levels at 60 min. By contrast, TSH levels were lowered by both doses of gastrin within 5 min of injection and the lowering persisted for 60 min. Intravenous injection of gastrin had no effect on plasma gonadotropin, GH and TSH, but induced an elevation in Prl levels. $\text{In}$$\text{vitro}$ incubation of hemipituitaries with gastrin failed to modify gonadotropin, GH or Prl but slightly inhibited TSH release at the highest dose of 5 μg gastrin. The results indicate that synthetic gastrin can alter pituitary hormone release in unrestrained OVX rats and implicate a hypothalamic site of action for the peptide to alter release of a gonadotropin, Prl and GH. Its effect on TSH release may be mediated both via hypothalamic neurons and by a direct action on pituitary thyrotrophs.     

Seasonal effects on female reproductive functions in the bovine (Indian breeds)

Reproductive function is mediated by season in the Indian breeds of cattle ($\text{Bos}$$\text{indicus}$). The reproductive endocrinology of $\text{Bos}$$\text{indicus}$ cattle differs from that of $\text{Bos}$$\text{taurus}$ breeds; the estrus is shorter and less intense and occurs late in relation to an estrogen stimulus. Moreover, the $\text{Bos}$$\text{indicus}$ female has a smaller preovulatory surge of luteinizing hormone (LH), which occurs earlier relative to the onset of estrus, and she ovulates sooner after the onset of estrus. The corpus luteum is smaller and contains less progesterone, and the serum progesterone concentration is lower in $\text{Bos}$$\text{indicus}$ females. Furthermore, they have fewer preovulatory LH surges than $\text{Bos}$$\text{taurus}$ females and their luteal cells are less responsive to LH in vitro during the winter. Their fertility is lower during the late fall and winter months. For $\text{Bos}$$\text{indicus}$ cattle, recovery of transferable embryos and survival of embryos in the recipient are at their maximum from July through October.     

The participation of corticosterone in luteinizing hormone releasing hormone (LH-RH) action on luteinizing hormone (LH) release from anterior pituitary cells in vitro

Corticosterone alone was not able to stimulate release of luteinizing hormone (LH) from anterior pituitary cells $\text{in}$$\text{vitro}$, but corticosterone in combination with luteinizing hormone releasing hormone (LHRH) augmented the release of LH into the culture media. These results may indicate that corticosterone may have the capacity to activate membrane receptors for LHRH in the gonadotrophs.     

Reproductive studies of Brahman cattle V. The effect of various dose levels of estradiol-17β upon serum luteinizing hormone in ovariectomized Brahman cows

Twelve mature chronically-ovariectomized Brahman cows were randomly assigned to receive three of six different dosages of estradiol-17_b (E2) at three different time periods such that at the termination of the trial six animals received each E2 dosage. The E2 was suspended in 0, 1, 2.5, 5, 10 and 20 milligrams. A two week period was maintained between injections. The cows were bled via coccygeal vessel puncture immediately before E2 injection, every 2 hr from 0 to 18 hr, every hr from 18 to 42 hr and every 2 hr from 42 to 48 hr postinjection. Blood was processed to yield serum and stored at ?20 Celsius. Serum luteinizing hormone (LH) was quantitated by validated radioimmunoassay. An LH surge was defined as a sustained elevation of LH at least two standard deviations above the level of LH prior to the rise and was observed in $\text{0}\text{6}$, $\text{3}\text{6}$, $\text{5}\text{6}$, $\text{5}\text{6}$, $\text{5}\text{6}$, and $\text{6}\text{6}$ cows administered 0, 1, 2.5, 5, 10, and 20 mg of E2, respectively. All animals injected with E2 responded with a significant initial decrease (independent of E2 dosage) in LH that persisted from 2 through 12 hr post E2 injection. No significant decrease in LH levels was recorded in animals injected with the corn oil vehicle. The time to the LH surge differed (P<.05) between 1 mg E2 (10 hr) vs 20 mg E2 (19.5 hr), 1 mg E2 vs 10 mg E2 (16.2 hr), and 2.5 mg E2 (12.4 hr) vs 20 mg estradiol-17_β. Luteinizing hormone concentrations at the onset of the surge did not differ (P>.10) between E2 dosages. The elapsed time from E2 injection to the peak LH value differed (P<.05) between 1 mg E2 (20.3 hr) vs 20 mg E2 (26.8 hr), and 2.5 mg E2 (21.2 hr) vs 20 mg estradiol-17_β. The peak LH value, the area under the LH curve and the duration of the LH surge did not differ (P>.10) with E2 dosage. The time to the end of the LH surge differed (P<.05) between 1 mg E2 (25.3 hr) vs 2.5 mg E2 (31.6 hr), 1 mg E2 vs 5 mg (34.4 hr), 1 mg E2 vs 10 mg E2 (34.8 hr), 1 mg E2 vs 20 mg E2 (37.3 hr), and 2.5 mg E2 vs 20 mg estradiol-17_β. Luteinizing hormone values at the termination of the surge did not differ (P>.10) between dosages nor did the LH values at the termination of the surge differ (P>.10) from LH concentrations observed at the onset of the LH surge.     

Prostaglandin levels in preovulatory follicles from rabbit ovaries perfused invitro

Prostaglandin (PG) levels in follicular fluid from preovulatory follicles of rabbit ovaries perfused $\text{in}$$\text{vitro}$ were measured in order to compare PG changes in this model system with those that occur $\text{in}$$\text{vivo}$ and in isolated, LH-treated follicles $\text{in}$$\text{barvitro}$. One ovary from each rabbit was perfused without further treatment (control). The other ovary was exposed to LH (0.1 or 1 ug/ml) beginning 1 hour (h) after initiation of perfusion. Samples of perfusion medium were taken at frequent intervals for measurement of PGE, PGF, progesterone and estradiol 17β. The perfusions were terminated when the first ovulation occurred or appeared imminent as judged by changes in the size and shape of the follicles. Follicular fluid was then rapidly aspirated from all large follicles on both ovaries for PGE and PGF measurement.Ovulations occurred only in the LH-treated ovaries. Progesterone and estradiol levels were significantly elevated in the perfusion medium within 1 h of LH treatment in comparison to controls. PG levels in perfusion medium from the control and LH-treated ovaries were not different throughout perfusion and increased in both groups. In contrast, PG levels measured in follicular fluid from LH-treated ovaries were 4- to 5-fold greater than in fluid from control ovaries. It is concluded that ovulation induced by LH in this experimental model is accompanied by an increase in follicular PG levels similar to that seen in other $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$ models. This difference in follicular PG levels between the LH-treated and control ovaries is, however, not reflected in the perfusion medium.     

The interaction between the heme c and heme d moieties of Pseudomonas nitrite reductase as revealed by magnetic and natural circular dichroism studies.

A possibility of a heme-heme interaction between the heme $\text{c}$ and heme $\text{d}$ moieties in $\text{Pseudomonas}$ nitrite reductase was examined by using magnetic and natural circular dichroism. The MCD of the heme $\text{c}$ moiety in the ferric enzyme was similar to that of mammalian ferricytochrome $\text{c}$ in shape and intensity, whereas in the reduced state the MCD intensity was considerably smaller than that of ferrocytochrome $\text{c}$. When the heme $\text{d}$ moiety was perturbed by the complex formation with CO, imidazole or cyanide as well as by pH changes, the depressed MCD was restored to the MCD level of mammalian ferrocytochrome $\text{c}$, accompanying conformational changes around the prosthetic groups. Thus, it was concluded that the heme-heme interaction exists only in the reduced enzyme and that this interaction is released under appropriate conditions.     

Stimulation by cyclic nucleotides of prostaglandin E production in isolated Graafian follicles

Rat Graafian follicles isolated intact responded to 8-Br-cyclic AMP and 8-Br-cyclic GMP with increased prostaglandin E (PGE) production during a 6 h incubation. By contrast, 8-Br-cyclic IMP, 8-Br-5′ AMP and 8-Br-5′ GMP were inactive in this respect. The effect of 8-Br-cyclic AMP and 8-Br-cyclic GMP was noted only after a lag period of about 4 h. Choleragen, LH, and the phosphodiesterase inhibitor (3-isobutyl-1-methyl-xanthine; IBMX) also stimulated PGE production. Actinomycin D and cycloheximide given simultaneously with 8-Br-cyclic AMP or LH prevented the stimulatory effect of these agents. Concomitant addition of arachidonic acid did not overcome the effect of these inhibitors.Administration of hCG $\text{in vivo}$ or incubation with LH $\text{in vitro}$ did not elevate endogenous ovarian free arachidonate, while PGE production was enhanced. Dexamethasone prevented this stimulatory effect of hCG.Collectively, the results suggest that stimulation of ovarian PGE production by cyclic mucleotides and LH is dependent on $\text{de novo}$ synthesis of one more components of the PG synthetase systme rather than on substrate availability. Cyclic nucleotides may mediate the stimulatory effect of gonadotropins on PGE production