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.     

Synthesis and biological properties of (D-Ala-6, des-Gly-NH2-10)-LH-RH ethylamide, a peptide with greatly enhanced LH- and FSH- releasing activity

A nonapeptide analog of luteinizing hormone-releasing hormone (LH-RH), [D-Ala⁶, des-Gly-NH₂¹⁰]-LH-RH ethylamide, was prepared by solid-phase methodology. The peptide was assayed against LH-RH in two $\text{in vivo}$ systems and was found to be many times more potent than the naturally occurring hormone. In one of the tests, based on elevation of LH and FSH levels after infusion into immature male rats, the analog showed LH-releasing activity of 1600% and FSH-releasing activity of 1200% compared to LH-RH.     

Human erythrocyte glucose 6-phosphate dehydrogenase. Content of bound coenzyme

A series of peptide analogs of luteinizing hormone releasing hormone (LH-RH), altered at position 6 and 10, was synthesized and evaluated $\text{in vivo}$ for the ability to induce ovulation in the diestrous rat and $\text{in vitro}$ for ability to release pituitary luteinizing hormone and follicle stimulating hormone. All the analogs with D-amino acid substitutions at position 6, even those with large bulky side chain, exhibited an amazingly high potency compared with the parent hormone, LH-RH. On the basis of the biological activities, structure-activity relationships in the central part of this molecule were discussed in detail.     

Effect of prolonged administration of small doses of LH-RH and LH-RH analogue [D-Ser (But)6] Des Gly-NH210 ethylamide on the release of LH and induction of ovulation in mid-anoestrous ewes

A natural process of LH release and induction of ovulation in anoestrous ewes was simulated by prolonged administration of small doses of LH-RH and its analogue [D-Ser(But)⁶] Des Gly-NH₂¹⁰ ethylamide. In the first series of experiments on 40 Merino ewes infusions of LH-RH were made into the maxillaris interna artery for 6 consecutive days for 6 h each day. Total doses of 24.0, 26.0, 28.0 and 32.0 μg per animal of varying and progressively increasing daily quantities of the hormone were administered in groups I, II, III and IV, respectively. In group V the animals were infused with a total dose of 28.0 μg LH-RH and injected additionally i.m. with 3.0 μg 17β-oestradiol on days 4 and 5 of the infusion of LH-RH. Ovulation did not occur earlier than days 4, 5 and 6 after the beginning of infusions. The highest number of positive reactions occurred in group IV ($\text{8}\text{10}$) and in group V ($\text{7}\text{8}$ animals). The pattern of LH peaks in general was correlated with the time of ovulations. The LH concentrations of the preovulatory peaks in experimental ewes were mostly lower than those in naturally ovulating animals. The corpora lutea were functional during the first 7 days after ovulation.In the second series of experiments on 26 Merino ewes the LH-RH analogue [D-Ser -(But)⁶] Des Gly-NH₂¹⁰ ethylamide was injected i.m. or i.a. for 6 consecutive days. Total doses of 15.5, 9.5 and 7.5 μg of the analogue per animal, administered at varying and progressively increasing daily doses in respective groups, induced several surges of LH in the same individuals for 2 or even 3 consecutive days. Corpora lutea and degenerating follicles in the form of cysts were found in the ovaries of animals of these groups. Very small daily doses ranging from 0.1 μg administered during the first 3 days, to 1.5 μg on day 5 of the treatment, released one surge of LH on day 5 of the treatment in all individuals with peaks ranging from 30.0 to 58.0 ng/ml and induction of ovulation with almost normal luteal function. On the basis of these experiments it is suggested that the evaluation of the effect of active substance (LH-RH or its analogue), its suitability and application of rightly chosen doses to induce the full physiological process of ovulation should be based not only on the release of LH and luteal function but also on tests of the ability of the released ovum to undergo fertilization and its further development.     

Independence of the differentiation of masculine and feminine sexual behavior in rats.

Male rats received Silastic implants of the aromatase inhibitor, 1,4,6-androstatriene-3, 17-dione (ATD), on days 2–10 of life. Controls received blank implants. There were no differences in the masculine sexual behavior of ATD and control males when they were tested as gonadally intact adults. In contrast, even without exogenous hormone treatment, nine of 14 ATD males exhibited lordosis behavior, whereas only one of 12 controls did so. In addition, during a sexual preference test in which access was provided to both a sexually receptive female and to a stud male, there was no difference in the proportions of ATD ($\text{11}\text{14}$) and control ($\text{7}\text{12}$) males that copulated with the stimulus female; however, seven of the ATD males also exhibited feminine sexual behavior including some instances of solicitation. Only one of the control males showed any lordosis behavior. In general, all animals spent more time with the stimulus female than with the stud male. At the termination of preference testing, all animals were castrated and then tested twice for feminine sexual behavior under exogenous estradiol benzoate and progesterone. All of the ATD males showed lordosis behavior with a mean lordosis quotient (LQ) of 85; and 11 of the 14 also showed solicitation behavior. Only five of 12 control males exhibited lordosis ($\text{X}\text{?}\text{LQ}\text{= 59}$) and only one showed solicitation behavior. These results indicate that the propensity of males to show feminine sexual behavior can be manipulated independently of the capacity for masculine sexual behavior. Moreover, our results suggest that the process of defeminization may occur primarily postnatally in rats since treatment during that period results in substantial increments in later feminine sexual behavior including solicitation behaviors.     

Production and characterization of an antiserum to synthetic gonadotropin-releasing hormone

The synthetic decapeptide “luteinizing hormone-releasing hormone” (LH-RH) was rendered antigenic by reaction of its histidine or tyrosine residues (7 : 3 approx.) with p-diazonium phenylacetic acid and coupling of the azo-derivatives formed to bovine serum albumin (BSA). Immunization of rabbits yielded antisera that bound ¹²⁵I-labeled LH-RH (approx. 50 pg) at dilutions up to 1:200, 000 and showed no cross-reaction with unrelated hypothalamic and pituitary hormones, extracts from rat cerebral cortex, and with small fragments of LH-RH. Cross-reaction was minimal (0.2%) with the free acid analogue of LH-RH, and moderate with $\text{des}$-pGlu LH-RH (20%), $\text{des}$-pGlu-His-LH-RH (2.4%) and with LH-RH analogues in which a single residue (No. 4–6 or No. 8) was exchanged by an amino-acid of similar character (1.2–12%). Biologically active hypothalamic extract and LH-RH produced parallel ¹²⁵I-LH-RH-binding inhibition curves, providing immunochemical support for the identity of the native releasing hormone with synthetic LH-RH.     

Dominance and aggression in social groups of male and female rats

Two experiments were performed to examine aggression and dominance in domestic male and female $\text{Rattus norvegicus}$ living in small mixed-sex (3 males and 3 females) groups. Experiment 1 examined the development of aggression in females. A single female (alpha) within each of the six colonies tested showed the preponderance of attacks on male intruders placed into the home-cage when male colony residents were absent. Over 12 weeks of intruder-aggression training female alphas showed only a mild nonsignificant elevation of aggressive behavior. A comparison of aggression of male and female colony alphas tested with opponents of each sex revealed that aggression was mainly directed at like-sex opponents, and that female attack was more defensive in character than male attack regardless of opponent sex. The highest intensity of aggression occurred when male alphas confronted male intruders. Although intruders never showed offense toward male residents, 61% of intruding males showed offense in response to attack by females.Experiment 2 investigated the relationship between aggressive dominance and competitive measures of dominance within each of 10 mixed-sex colonies. Alpha stat s of male and female colony residents did not reliably predict priority of access to food or water in tests of direct resource competition with like-sex colony members. When colony males were simultaneously tested for copulation, the copulatory behavior of alpha males was significantly greater than that of other colony males. Results are discussed in relation to the role of aggression in the reproductive strategy of male and female $\text{Rattus norvegicus}$.     

Basal and LH-RH-stimulated gonadotropin release after transport stress in male rats

A total of 120 male rats of the Sprague-Dawley-strain (6 weeks old) were used in this experiment. 5 groups of 12 animals each were treated intraperitoneally with 200 ng gonadotropin releasing hormone (LH-RH) per animal. 30 minutes later blood was sampled by heart puncture. Group I were animals without transport, group II immediately after, group III one day, group IV one week and group V six weeks after a standardised transport. Another 5 groups were subjected to the same protocol but received saline i.p. instead of LH-RH. Serum levels of LH and FSH were estimated by radioimmunoassay. LH and FSH serum levels could be stimulated by LH-RH in all groups. A significant rise of basal and LH-RH stimulated LH levels was observed until the first day after transport. Thereafter a drop was registered. No consistent patterns of basal as well LH-RH stimulated FSH-levels were noted. These data combine to suggest an elevation of LH-RH secretion as response to the stress. This results in a sensibilisation of the pituitary to exogenous LH-RH.     

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.     

Synthesis and biological activities of analogs of luteinizing hormone-releasing hormone (LH-RH) substituted in position 1 or 2

Syntheses are described of [Pro¹]-LH-RH, [Orotic acid¹]-LH-RH, [Glu¹]-LH-RH, [Ser²]-LH-RH, [Leu²]-LH-RH, [Gln²]-LH-RH and [Phe²]-LH-RH. The LH-releasing hormone (LH-RH) activity of each of these peptides was compared with that of natural LH-RH $\text{in vivo}$. [Glu¹]-LH-RH and [Phe²]-LH-RH had significant LH-RH activity, while all the other analogs possessed extremely low activities. These findings are briefly discussed in the light of the structure-activity relationship for LH-RH.     

Effect of bromocriptine on the hypothalamo-pituitary function in adult male rats.

Daily oral administration of bromocriptine (50 μg/kg) to adult male rats, suppressed serum prolactin levels. The pituitary prolactin levels remained unaltered. Serum FSH levels as well as pituitary FSH levels showed no significant change as compared to the controls. Serum LH levels were significantly decreased in spite of the high pituitary LH levels, in bromocriptine treated rats. In the drug treated rats, $\text{in vitro}$ sensitivity of the pituitary to the exogenous LH-RH was not altered; whereas hypothalamic LH-RH content was considerably lowered. These observations suggest the possible effect of bromocriptine on the synthesis of LH-RH in the hypothalamus which leads to the accumulation of LH in the pituitary and decline of serum LH.     

Reduction of LH-RH pituitary and estradiol uterine binding sites by a superactive analog of luteinizing hormone-releasing hormone

The ability of the Luteinizing Hormone-Releasing Hormone (LH-RH) analogs to displace LH-RH from its pituitary receptors was evaluated $\text{in}\text{vitro}$. The two superactive analogs tested showed higher potency than the antagonists and LH-RH itself, D-Trp⁶-LH-RH being the most potent. The LH-RH specific binding activity in the pituitary fluctuated throughout the age of the rats. The highest number of LH-RH binding sites were seen on day 35 of age (276 fmol × 10^?2/pit) and an increment was induced by 0.05 μg D-Trp⁶-LH-RH (400 fmol × 10^?2/pit). However, 1 μg D-Trp⁶-LH-RH reduced the binding of LH-RH at all the times studied. In the control animals the number of estradiol binding sites increased on day 42 of age, and 0.05 μg D-Trp⁶-LH-RH augmented them on day 35 of age. On the contrary, 1 μg D-Trp⁶-LH-RH diminished the estradiol uterine receptors at all the times studied. Similar results were obtained in the ovariectomized-hypophysectomized rats on day 35 of age. Our studies demonstrated a biphasic action of D-Trp⁶-LH-RH on LH-RH pituitary receptors and a direct effect on uterus which could be mediated through the uterine estradiol receptors.     

Differences in the degradation of hypothalamic releasing factors by rat and human serum

Rat serum is 2–5 fold more active than human serum in cleaving the three hypothalamic releasing hormones, luteinizing hormone releasing hormone (LH-RH), thyrotropin releasing hormone (TRH), and somatostatin. LH-RH was degraded by two distinct enzymatic mechanisms; 1) endopeptidase cleavage, 2) C-terminal cleavage. The C-terminal cleaving enzyme was active in rat serum but present only in trace levels in human. These mechanisms were substantiated by the use of suitably substituted analogs; D-Ala at position 6 of LH-RH prevented cleavage at the -Tyr⁵-D-Ala⁶-Leu⁷-site and the presence of ethylamide (C₂H₅NH₂) at position 10 inhibited significantly the action of the second enzyme. These analogs have an enhanced biological activity $\text{in}$$\text{vivo}$ which correlates well with their decreased rate of degradation. Somatostatin was degraded by endopeptidase cleavage at one or more sites. D-Trip in position 8 blocked cleavage of the -Trp⁸-Lys⁹-bond, reducing significantly the rate of degradation. This also correlates well with the enhanced biological potency of the (D-Trp⁸)-somatostatin analog. TRH was degraded by cleavage of the pyroGlu-His and His-Pro.NH₂ bonds with the release of free His and Pro. The analog (3-Me-His²)-TRH was degraded by a similar mechanism with the release of 3-Me-His.     

Onset of the receptive and proceptive components of feminine sexual behavior in rats following the intravenous administration of progesterone

The present study was carried out in order to assess the time course of action of progesterone (P) in the facilitation of complete feminine sexual behavior. Female rats (estrogen primed via 5% E₂ Silastic capsules) were given 200 μg of P either intravenously (iv) or subcutaneously (sc), and tested for estrous behavior at $\text{1}\text{4}$, $\text{1}\text{2}$, 1, 2, and 4 hr after treatment. Among iv-treated animals, significant amounts of lordosis behavior were seen as early as $\text{1}\text{2}$ hr, and a dramatic rise in solicitation behavior was observed at 2 hr. Although sc-treated animals displayed significant amounts of lordosis and solicitation behavior at 2 hr, the behavior was not maximal until 4 hr. Intravenous administration of 400 μg P was equipotent to 200 μg P, whereas 50 μg of iv P was relatively ineffective. A dual mechanism hypothesis pertaining to progesterone's actions in the facilitation of both the receptive and preceptive components of feminine sexual behavior in rats is discussed.     

Karyotypic normalcy and quasi-normalcy of developmentally totipotent mouse teratocarcinoma cells

Malignant mouse teratocarcinoma cells are, in some cases, able to undergo normal, complete differentiation after injection into blastocysts. Thus far, only three lines—of unrelated origin—have been found (all in this laboratory) to be developmentally totipotent in blastocyst tests. The karyotypes of these lines, and their somatic- and germ-cell derivatives, were investigated by G-banding methods, as a possible clue to their developmental superiority. The first, OTT 6050 (129 strain), is an embryo-derived induced tumor maintained as an ascites transplant line. Its stem cells (from embryoid body “cores”) have 40 chromosomes in the modal class, which comprises two subclasses: one all normal and one with a metacentric chromosome (isochromosome-8). However, mosaic animals from injected blastocysts have only the normal subclass in their teratocarcinomaderived cells; all are of $\text{X}\text{Y}$ male sex chromosome type. Presence of the Y chromosome was verified after transmission through the germ line of two fertile mosaic males, in their F₁ male progeny. The second teratocarcinoma line, 72484-395 (LT strain), is a spontaneous ovarian solid tumor maintained by subcutaneous transplantation. Karyotypes of cells from the tumor, and also of teratocarcinoma-derived cells in mosaic animals, were normal and of $\text{X}\text{X}$ female sex chromosome type. Karyotypes of the F₁ progeny, from tumor-strain germ cells of a fertile mosaic female, were also normal. The third line, NG 2 (129 strain), is a mutant clonal in vitro line deficient in hypoxanthine phosphoribosyltransferase. It originated from an embryo-derived experimental tumor (OTT 5568) that was established in culture (PSA1 line); the culture was then mutagenized and selected for 6-thioguanine resistance. The NG 2 line proved to be quasi-normal, with only two karyotypic anomalies: trisomy of chromosome 6 and $\text{X}\text{O}$ female sex chromosome constitution. Thus, developmental totipotency in all three lines, including one maintained in vitro, is accompanied by karyotypic normalcy or near-normalcy. Other culture lines reported to be aneuploid have not yet given evidence of totipotency. Karyotypic normalcy may therefore have predictive value useful in choosing teratocarcinoma lines with relatively high developmental prospects. This is of importance in identifying those mutant lines that would be promising candidates for introduction, via blastocyst injection, of specific mutant genes into mice.     

Two sex pheromone components of the tobacco budworm moth, Heliothis virescens

Two compounds were isolated from female $\text{Heliothis}$$\text{virescens}$ (Lepidoptera: Noctuidae) extracts and identified as $\text{cis}$-9-tetradecenal and $\text{cis}$-ll-hexadecenal. Together they elicit intense male $\text{H}$. $\text{virescnes}$ response in laboratory tests and have attracted males in the field. Although $\text{cis}$-ll-hexadecenal is an $\text{H}$. $\text{zea}$ sex pheromone, no evidence was obtained for $\text{cis}$-9-tetradecenal in $\text{H}$. $\text{zea}$.     

Control of estrogen and androgen receptors in the rat pineal gland by catecholamine transmitter

Sucrose gradient studies of rat pineal cytosol incubated with ³H-estradiol (female pineals) or ³H-5 α -dihydrotestosterone (male pineals) revealed a radioactivity peak in the 8 S region which disappeared after superior cervical ganglionectomy or incubation with excess unlabeled hormone. Ganglionectomy decreased significantly estradiol and testosterone uptake by the pineal gland $\text{in vitro}$ as well as high affinity binding to pineal cytoplasmic and nuclear components. Norepinephrine treatment counteracted all the effects of ganglionectomy but was unable to modify hormone uptake and binding by the pineal gland of sham-operated controls. Pre-treatment with actinomycin D or propranolol but not with phentolamine impaired norepinephrine effects; propranolol blockage however was only partial. Administration of isoproterenol, L-dopa or phentolamine increased hormone uptake by denervated pineals. The effects of isoproterenol were also observed $\text{in vitro}$ and were blocked by propranolol. These results indicate that sex steroid receptors in the pinealocytes are controlled by norepinephrine via beta-adrenergic receptors and that depletion of neural norepinephrine enhanced responsiveness of pineal hormone receptors to exogenous catecholamines.     

Hyperprolactinemia preferentially inhibits erectile function in adrenalectomized male rats

To determine if the inhibitory effects of hyperprolactinemia on sexual arousal and serum LH levels could be dissociated from those on erectile function, copulatory behavior was examined in pituitary-grafted, adrenalectomized male rats that had been castrated and given 20mm subcutaneous testosterone implants. Whereas transplantation of three pituitaries under the kidney capsules inhibited mounting rates in intact animals, pituitary grafting did not significantly reduce mounting rates in the adrenalectomized group beyond the effect of adrenalectomy alone. In contrast, the effects of pituitary grafting on erectile function were enhanced in the adrenalectomized animals. Hyperprolactinemia also caused a significant reduction in serum LH, but only in the intact animals. These results suggest that: 1. the effects of hyperprolactinemia on erectile function occur independently from those on sexual arousal, and 2. the inhibitory effects of hyperprolactinemia on sexual arousal are linked to the effects of hyperprolactinemia on LH release.     

Centrally applied somatostatin influences morphology of pituitary FSH cells but not FSH release

Effects of i.c.v. administered somatostatins on morphology and function of pituitary FSH cells were examined in adult male Wistar rats. The animals were given three 1 microg doses of SRIH-14 or SRIH-28 in 5 microl saline every second day. Controls were given the same volume of saline only. Both SRIH treatments lead to a significant decrease in absolute pituitary weight and volume of FSH cells in comparison with controls. Relative pituitary weight was significantly decreased only after SRIH-28 treatments, while FSH secretion was insignificantly decreased by both SRIH treatments. Our results indicate that i.c.v. applied somatostatins have significant inhibitory effect on absolute pituitary weight and on the volume of FSH cells, without affecting the hormone secretion in male rats.     

Alteration in the normal pattern of serum testosterone and 5α-androstane-3α,17β-diol in the immature male rat following chronic treatment with luteinizing hormone releasing hormone or luteinizing hormone

A major component of sexual maturation in the male rat is a progressive decline in serum concentrations of 5α-androstane-3α,17β-diol (3α-diol) and a concomitant increase in testicular testosterone biosynthesis and secretion. Chronic administration of synthetic luteinizing hormone releasing hormone (LHRH) or luteinizing hormone (LH)/human chorionic gonadotropin (hCG) to immature male rats has been shown to result in a delay in sexual maturation as evidenced by decreased sex accessory gland weights and altered testicular testosterone production. We have examined the postulate that such treatments may either reverse or retard the normal developmental pattern of serum testosterone and 3α-diol concentrations. Chronic $\text{in vivo}$ treatment of 28 day old immature male rats for 2 weeks with daily injections of either 0.5 μg of LHRH, 1.0 μg of LHRH, or 30 μg of LH was found to result in significant reductions in weights of the seminal vesicles and ventral prostate glands and diminutions in serum testosterone concentrations. Serum content of 3α-diol was either unchanged or slightly elevated in the LHRH treated animals and increased significantly in the LH treated animals. These data suggest that either a reversal of or retardation in the normal developmental pattern of serum testosterone and 3α-diol content has been achieved in the immature male rat by chronic LHRH or LH treatment.