Normal Immature Rats Research Articles

18F–labeled estradiol derivative for targeting estrogen receptor-expressing breast cancer

IntroductionA novel radiotracer 1‑(2‑(2‑(2‑[18F]fluoroethoxy)ethoxy)ethyl)‑1H‑1,2,3‑triazole‑estradiol ([18F]FETE) was successfully synthesized, characterized and evaluated in mice for estrogen receptor (ER)-positive breast cancer targeting with positron emission tomography (PET) imaging. MethodsThe tosylate precursor 3 was radiolabeled with 18F and then reacted with 17α‑ethinyl‑estradiol to produce the final [18F]FETE. The physicochemical properties of [18F]FETE were tested in vitro, including determination of the octanol/water partition coefficient, stability and cellular uptake in MCF-7 (ER-positive) and MDA-MB-231 (ER-negative) cells. An ex vivo biodistribution study was performed in normal Sprague Dawley rats, and in vivo microPET imaging was performed on MCF-7 and MDA-MB-231 tumor-bearing mice. The results of biodistribution and PET imaging of [18F]FETE were compared with that of known 16α‑[18F]fuoro‑17β‑estradiol ([18F]FES). Radiation dose estimates for [18F]FETE were also analyzed. Results[18F]FETE was obtained in high radiochemical yield (46.59 ± 8.06%) with high radiochemical purity (>99%) after HPLC purification and high molar activity (15.45 ± 3.15 GBq/μmol). [18F]FETE is a moderate lipophilic compound with good in vitro stability and the total synthesis time was 55 to 65 min. In biodistribution studies, [18F]FETE showed high uptake in the ER-abundant uterine tissue of normal immature SD rats (8.55 ± 1.21 and 6.83 ± 1.70%ID/g at 1 h after intravenous and intraperitoneal injection, respectively), and could be blocked with estradiol effectively (the uterus uptake was decreased to 0.63 ± 0.35%ID/g at 1 h after iv injection). MicroPET imaging of tumor-bearing mice with [18F]FETE at 1 h after iv injection revealed considerable uptake in ER-positive MCF-7 tumors (4.63 ± 0.73%ID/g) that could be inhibited (1.47 ± 0.29%ID/g) and low uptake in ER-negative MDA-MB-231 tumors (1.97 ± 0.36%ID/g). [18F]FES has relatively low uptake in ER-positive tumor (0.24 ± 0.19%ID/g) when compared with [18F]FETE. The adult female effective radiation dose of [18F]FETE in mice was estimated as 0.0022 mSv/MBq. ConclusionsA novel 17α‑ethinyl‑estradiol-based ER probe [18F]FETE was developed with high molar activity and good in vitro stability. Based on the results of bio-evaluation in normal immature rats and tumor-bearing mice, it might be a promising candidate for specific PET imaging of ER-positive breast cancer.

Electroporation-induced changes in normal immature rat myoblasts (H9C2)

Application of a high electric field causes an electric shock to the heart. This is utilized in defibrillation to reestablish normal contraction rhythms during dangerous arrhythmias or in cardiac arrest. If shock-induced transmembrane potentials are large enough, they can cause tissue destruction due to irreversible electroporation (EP). Also electrochemotherapy of nearby tissues may have an adverse effect on the heart. Herein, we present experimental data on effects of electroporation in culture of cardiac cells (H9C2). The electric field was applied in short pulses of 25-3250 V/cm, 50 µs each. The viability of cells was tested by MTT assay after 24 hours. For detection of DNA fragmentation, associated with apoptosis, alkaline and neutral comet assays were performed after EP. Additionally phase contrast images of cells obtained directly after EP were analyzed. Although cell images indicated disruption of cell membranes after EP with high intensities, only a few percent of apoptotic cells and no necrotic effects in the cell nucleus could be observed in comet assay tests performed 2 hours post EP. MTT viability test showed that pulse intensities above 375 V/cm are destructive for myocytes viability.

Reproductive effects of Ficus asperifolia (Moraceae) in female rats.

The reproductive effects of Ficus asperifolia in female rats were investigated in the present study. Sperm-positive adult female rats were orally administered (P.O.) either the aqueous and methanol extracts of Ficus asperifolia (100 and 500mg/kg), distilled water (10ml/kg) or 5%Tween 80 (10ml/kg) for seven days. On day 10 of pregnancy, the implantation sites were recorded. In the fertility study, adult female rats received the same test substances for 21 days and, the fertility index and litter size determined. In the uterotrophic test, normal and ovariectomized immature rats were treated for seven days with the dry extract of Ficus asperifolia (100 and 500mg/kg) in the absence and presence of 17ȃ-estradiol benzoate 1µg/animal/day, s.c. On day 8, the uterine growth index was measured. Results of the study showed a significant increase (p<0.05) in the implantation sites and litter size of animals receiving 100mg/kg of the aqueous extract of Ficus asperifolia. In the estrogenic assay, normal immature rats were sensitive to the treatment with Ficus asperifolia than the ovariectomized ones. Our results give added scientific support to the popular use of Ficus asperifolia in the treatment of some cases of women's sterility/infertility related problems.

Donor Sertoli cells transplanted into irradiated rat testes stimulate partial recovery of endogenous spermatogenesis

Irradiation of rat testes leads to the failure to support differentiation of the surviving spermatogonia due to damage of the somatic environment. To determine the involvement of Sertoli cells in this somatic damage, we transplanted seminiferous tubule cells from normal immature GFP-transgenic rats into the testes of irradiated rats. The donor Sertoli cells colonized and developed in the host testes. In many seminiferous tubules, the donor Sertoli cells formed abnormal spherical structures in the lumen, but in some tubules they formed a normal-appearing epithelium, but with only isolated spermatogonia, on the basement membrane. When the donor cells were injected into the interstitial region of the testis, they formed tubule-like structures containing Sertoli cells and occasional isolated spermatogonia, both of donor origin. Surprisingly, in host tubules adjacent to these newly formed donor-cell tubules or adjacent to the endogenous tubules with abnormal donor Sertoli-cell structures, endogenous spermatogonia differentiated to the spermatocyte or even to spermatid stages. Around these newly donor cell-formed tubules and the host tubules with abnormal donor Sertoli-cell structures, many cells including macrophages, which perhaps represented chronic inflammation, accumulated in the interstitium. We conclude that the donor Sertoli cells that colonized the seminiferous tubules did not directly support recovery of spermatogenesis. Instead, the colonizing Sertoli cells acted indirectly on the interstitium to stimulate localized differentiation of endogenous spermatogonia.

Nerve excitability properties in Charcot-Marie-Tooth disease type 1A.

Charcot-Marie-Tooth disease type 1A (CMT1A) is commonly considered a prototype of a hereditary demyelinating polyneuropathy. Apart from the myelin involvement, there has been little information on axonal membrane properties in this condition. Taking advantage of the uniform nature of the disease process, we undertook the in vivo assessment of multiple axonal excitability properties at the median nerve in nine CMT1A patients with PMP22 (peripheral myelin protein 22) gene duplication and 53 controls. The thresholds of CMT1A patients were much higher than normal, and threshold electrotonus (TE) exhibited a consistent pattern of abnormalities: early steep changes (fanning out) of both hyperpolarizing and depolarizing responses were followed by increased inward rectification to hyperpolarizing currents and unusually fast accommodation to depolarizing currents. Strength-duration time constants and the shapes of recovery cycles were normal, although refractoriness and superexcitability were reduced relative to controls. The high thresholds and early fanning out of electrotonus indicated altered cable properties, such that a greater proportion than normal of applied currents reached internodal rather than nodal axolemma. The rapid accommodation to depolarizing currents suggested activation of fast K+ channels, which are normally sequestered from the nodal membrane. The excitability abnormalities are therefore consistent with a demyelinating pathology and exposure or spread of K+ channels from under the myelin. It remains to be seen whether the TE abnormalities in CMT1A, which resemble previous recordings from normal immature rats, can be distinguished from those in acquired demyelinating neuropathies.

Hippocampal sclerosis revisited

Studies dating back more than 150 years reported a relationship between hippocampal sclerosis and epilepsy. Retrospective studies of patients who underwent temporal lobectomy for intractable partial epilepsy found a relationship between a history of early childhood convulsions, hippocampal sclerosis, and the development of temporal lobe epilepsy. Many believe that febrile seizures lead to hippocampal damage and this in turn predisposes the patient to the development of temporal lobe epilepsy. Studies in adult rats have shown that seizures can lead to hippocampal damage and unprovoked recurrent seizures. However, many questions remain as to the relevance of early childhood seizures to hippocampal sclerosis and temporal lobe epilepsy. Human prospective epidemiologic studies have not shown a relationship between early childhood seizures and temporal lobe epilepsy. Recent MRI studies in humans suggest that a preexisting hippocampal lesion may predispose infants to experience febrile seizures, later on hippocampal sclerosis, and possibly temporal lobe epilepsy may occur. Unlike the studies in adult rats, normal immature rats with seizures have not been shown to develop hippocampal damage or unprovoked seizures in adulthood. Furthermore, animal studies reveal that preexisting brain abnormalities can predispose to hippocampal damage following seizures early in life. This paper reviews evidence for and against the view that early childhood convulsions, hippocampal sclerosis, and temporal lobe epilepsy are related, while also exploring clinical and animal studies on how seizures can lead to hippocampal damage, and how this can result in temporal lobe epilepsy. By better understanding the cause and effect relationship between early childhood seizures and hippocampal injury in normal and abnormal brains specific treatments can be developed that target the pathogenesis of epilepsy.

Immunohistochemical localization of androgen receptors in the rat testis: evidence for stage-dependent expression and regulation by androgens.

Androgens are essential for the maintenance of normal spermatogenesis in the rat. We assessed the sites, developmental pattern, and hormonal control of androgen receptors (AR) in the rat testis. Adult male rats were studied after 1) no treatment; 2) ethane dimethane sulfonate (EDS), which eradicates Leydig cells and endogenous testosterone (T); 3) EDS plus T replacement beginning at the time of EDS administration; or 4) methoxyacetic acid, which leads to the loss of specific germ cell types. Testes were also obtained from normal immature rats (aged 5, 14, 16, 21, 28, 31, 35, 38, and 45 days). After microwave antigen retrieval, immunohistochemistry was performed using a rabbit polyclonal antibody (Novocastra) raised against a peptide unique to the N-terminal region of the AR and detection with biotinylated swine antirabbit immunoglobulin G, avidin-biotin complex/alkaline phosphatase, and nitroblue tetrazolium salt (NBT)/5 bromo-4-chloro-3-indolylphosphate (BCIP) substrate. In adults, nuclear immunostaining of Sertoli cells (SC) increased progressively in intensity from stages II through VII of the spermatogenic cycle, and then declined precipitously during stage VIII to become barely detectable in stages IX-XIII. Prominent AR immunostaining was also evident in peritubular myoid cells, arterioles, and interstitial cells; staining in these cells did not vary with the stage of the cycle of the adjacent tubules. EDS caused a severe loss of AR immunostaining in all cell types. Replacement of T in EDS-treated animals resulted in a pattern of AR immunostaining comparable to that in controls, although staining intensity was reduced. Methoxyacetic acid administration did not affect the pattern of AR staining. In immature rats, peritubular myoid cell immunostaining was prominent from day 5; SC staining was detectable on day 5, increased in intensity with age, and became stage dependent between days 21-35. The following conclusions were reached. 1) Immunohistochemically detectable AR expression in SC occurs predominantly in stages II-VII of the spermatogenic cycle, with highest levels at stage VII. 2) AR immunostaining is also prominent in peritubular myoid cells, arterioles, and Leydig cells (but not in germ cells), but is unrelated to the stage of adjacent tubules. 3) Endogenous T and/or its metabolites control the expression of AR in the testis. 4) AR immunostaining is detectable by day 5 of age and becomes stage specific in SC between days 21-35.

Pituitary and Gonadal Hormone-Dependent and - Independent Induction of Follicle-Stimulating Hormone Receptors in the Developing Testis

The number of FSH receptors increases during testicular development in several species of mammals. Hypophysectomy and hormonal replacement were performed to identify the factors that induce developmental changes in testicular FSH receptors. Male rats of the Wistar/Tw strain were hypophysectomized at 9, 16, 23, or 30 days or 3 months of age. These rats were killed 10 days after surgery along with intact control rats, and the testes were removed for receptor assay. A group of intact rats was killed on the day of surgery as initial controls. All of the hypophysectomized immature rats showed a higher density of FSH binding (FSH binding per unit weight) and a lower total FSH binding (FSH binding per two testes) compared with each of the matched intact control rats. In contrast to FSH binding, not only the total LH binding but also the density of LH binding were invariably lower in the hypophysectomized rats than in the intact control rats. Unlike hypophysectomy at other immature ages, surgery at 9 days of age was followed by significant increases in testicular weight, density of FSH binding, and total FSH binding compared with those values in the initial control rats. There was no significant effect of hypophysectomy on total FSH binding in adult animals, in contrast to the marked decrease in total LH binding. When male rats hypophysectomized at 25 days of age were injected with FSH for 5 days beginning on the 11th day after surgery, dose-dependent increases in total FSH binding and testicular weight were observed. Testosterone treatment induced an increase only in the total FSH binding, but its effect was less potent. Scatchard plot analyses of the binding suggested that changes in FSH binding with age, after hypophysectomy, and after hormonal administration were due to changes in the number of binding sites. Plasma FSH concentrations in all postoperative rats were below or at the level of detectability, indicating that hypophysectomy was successful. In normal immature rats, a significant increase in the plasma FSH level was detected only from 9-19 days of age, in contrast to the continuous increase in total FSH binding during testicular development. These results suggest that FSH and testosterone act as hormonal factors to induce an increase in the number of FSH receptors in the developing testis. Other factors that are independent of pituitary and sex hormones may also contribute to FSH receptor induction.

The Production of Transforming Growth Factor-β Activity by Rat Granulosa Cell Cultures*

We have examined whether granulosa cells (GC) secrete transforming growth factor-beta (TGF beta)-like activity using cell cultures prepared from diethylstilbestrol-primed female rats. Our results indicate that a significant level of active as well as latent TGF beta activity is found in defined GC culture medium as assessed by 1) potentiation of FSH-induced differentiation of rat GC, 2) neutralization of its activity by anti-TGF beta immunoglobulin, 3) inhibition of DNA synthesis in mink lung epithelial cells (CCl 64), and 4) activation of latent TGF beta activity by either acid or heat treatment. TGF beta production was more pronounced when the cells were seeded on fibronectin-coated plates. There was no difference in the level of TGF beta secretion by GC preparations derived from either diethylstilbestrol-primed immature or normal immature rats or adult rats. Furthermore, rat GC-conditioned medium contained much more TGF beta activity than medium from normal rat kidney cells (NRK 49-F), human prostatic adenocarcinoma cells (PC-3), or porcine GC. Rat thecal/interstitial cell culture medium contained activity comparable to that of GC medium. We conclude that rat GC preparations secrete a high level of TGF beta activity in vitro. Taken together with previous results, this indicates the possibility that TGF beta may be an autocrine regulator as well as a paracrine one within the ovarian follicle. Moreover, because of the high level of TGF beta activity produced, the rat GC culture system appears to be a useful experimental model for further exploring relationships between TGF beta production and its action.

Induction of ovulation, mating, and conception in androgen-treated immature rats.

Attempts were made to induce pregnancy in androgen-treated immature rats. Treatment with PMSG alone, which causes ovulation in normal immature rats, failed to cause ovulation in androgenized rats. However, treatment with PMSG plus LHRH was effective in causing ovulation. After ovulation, some of the normal and androgenized rats mated. Normal mated rats became pregnant but androgenized mated rats did not. However, when a pituitary gland was transplanted from a normal rat into the kidney capsule of an androgenized rat to maintain functional corpora lutea, implantation occurred in some of the mated animals. The positive decidual reaction in the uteri of such androgenized rats was similar to that observed in normal rats. These results suggest that the uterine sensitivity to blastocyst implantation of androgenized immature rats may be normal.

A histometrical study of immunohistochemically identified mitotic adenohypophysial cells in immature and mature castrated rats.

Two weeks after operation the effects of castration on all types of mitotic adenohypophysial cells in 30-day-old (immature) and 75-day-old (mature) male rats were studied immunohistochemically by light microscopy. The total number of mitotic cells in gland specimens increased 1.4 times in immature rats, but remained unchanged in mature animals; mitotic activity (mitotic cell number/mm2) was not affected in either age group. The population of mitotic gonadotrophs increased five to six times in comparison with intact controls. In addition, the percentage of mitotic gonadotrophs and their mitotic activity were raised in both age groups. Castration affected the mitosis of prolactin cells. The percentage of mitotic prolactin cells was reduced by one-third in castrated immature rats, but the total number and percentage of mitotic prolactin cells, as well as their mitotic activity, was reduced by about two-thirds in mature castrated rats. No significant changes in mitotic ACTH, TSH, GH and immunonegative cells were found after castration in either group of animals. Mitotic cells were more numerous in the anterior than in the posterior region of the gland in normal (uncastrated) immature rats, but were uniformly scattered in both regions in normal mature rats. On the other hand, castration induced a high population of mitotic cells in the anterior region regardless of age. This high population was the result of the enhanced mitosis of gonadotrophs. It is concluded that 2 weeks after castration the increment of mitotic divisions of all types of pituitary cells is age-dependent, and that mitosis takes place frequently in gonadotrophs and less frequently in prolactin cells.

A high-affinity binding site for the antioestrogens, tamoxifen and CI 628, in immature rat uterine cytosol which is distinct from the oestrogen receptor.

A high-affinity, saturable antioestrogen binding site, which does not bind oestradiol, has been reported to exist in a number of oestrogen target tissues but not in the immature rat uterus. This study reports the results of a more thorough search for this site in immature rat uterine cytosol. When concentrations of uterine cytoplasmic receptor were selectively depleted by translocation of 90-95% of the cytoplasmic oestrogen receptor to the nucleus, saturation analysis studies revealed that the antioestrogens, tamoxifen and CI 628, were bound to high-affinity, saturable binding sites which were present at about 2.5 times the concentration of the residual oestrogen receptor sites. Oestradiol could only partially inhibit the binding of tritiated antioestrogens to their saturable binding sites in this material indicating that a significant proportion of these sites were distinct from the oestrogen receptor sites. This was confirmed in experiments where oestrogen receptor sites were saturated in vitro with oestradiol and high-affinity, saturable sites for CI 628 and tamoxifen were still present. The CI 628 and tamoxifen had high affinity for these sites with dissociation constants of 1.0-1.6 nmol/l. These specific antioestrogen binding sites were present at about 5% of the concentration of oestrogen receptors in normal immature rat uterine cytosol which probably explains their previous lack of detections in this material.

Effects of differential environments on plasticity of dendrites of cortical pyramidal neurons in adult rats

Thickening of the frontal cortex and especially the occipital cortex was observed in adult rats after exposure to the “enriched” condition. An increase in branching and in length of terminal segments was found in the dendritic tree of pyramidal cells in layers II and III of the visual cortex of the adult rat after exposure to “standard” and enriched conditions. These exposures began at day 112 and continued 30 days. The increase observed in the basal dendritic tree of pyramidal cells in the superficial layers was significantly greater in the enriched conditions than in the standard condition. It appeared, furthermore, that branching occurred predominantly on basal terminal segments of all orders at a considerable distance from the tip. This mode of growth is similar to that observed in the cortex of normal immature rats. The differential conditions did not influence the bifurcation angles. The dendritic and cortical changes and changes reported in the literature indicate that the effects of differential experience are not limited to a short, “critical” period.

Effect of age of the rat on induction of acetylcholinesterase of the brain by 17β-estradiol

1. 1. The activity of acetylcholinesterase and its induction by estradiol in the cerebral hemisphere and cerebellum of immature (9-), adult (29-) and old (65-week) female rats were studied to understand the mechanism of aging. 2. 2. The activity of the enzyme of the cerebral hemisphere is highest in the immature rat and decreases thereafter. There is no such change in its activity in the cerebellum as a function of age. 3. 3. Estradiol increases the activity of the enzyme in the cerebral hemisphere and cerebellum of normal immature and adult rats. The magnitude of stimulation is higher in immature rats. Estradiol induces the enzyme in both the organs of ovariectomized immature and adult rats, but not of old rats. The induction of the enzyme by the hormone is actinomycin D sensitive. 4. 4. Kinetic, electrophoretic and immunological studies on the partially purified enzyme indicate the presence of a single molecular species of acetylcholinesterase at all the ages studied.

SUPEROVULATION IN THE ANDROGEN-TREATED IMMATURE RAT.

Journal Article Superovulation in the Androgen-Treated Immature Rat Get access K. BROWN-GRANT, K. BROWN-GRANT 1tDepartment of Human Anatomy, University of Oxford, England, and Department of Biological Sciences, Purdue University Lafayette, Indiana Search for other works by this author on: Oxford Academic Google Scholar D. L. QUINN, D. L. QUINN 1tDepartment of Human Anatomy, University of Oxford, England, and Department of Biological Sciences, Purdue University Lafayette, Indiana Search for other works by this author on: Oxford Academic Google Scholar M. X. ZARROW M. X. ZARROW 1tDepartment of Human Anatomy, University of Oxford, England, and Department of Biological Sciences, Purdue University Lafayette, Indiana Search for other works by this author on: Oxford Academic Google Scholar Endocrinology, Volume 74, Issue 5, 1 May 1964, Pages 811–813, https://doi.org/10.1210/endo-74-5-811 Published: 01 May 1964 Article history Received: 18 November 1963 Published: 01 May 1964

ENDOGENOUS GONADOTROPIC ACTIVITY IN MARE SERUM SUBSEQUENT TO CHRONIC TREATMENT WITH GONADOTROPIN.

Over 100 samples of sera from 2 mares chronically treated with HCG alone or combined with FSH as an antigen were tested for gonadotropic activity in normal immature female rats. The sera were also tested in normal immature males and in hypophysectomized male and female rats. An FSH-like response was obtained in the normal immature female rat with the sera of the mare treated with HCG alone for about a 6-month period after cessation of treatment with antigen. In the mare receiving the sequential treatment with HCG followed by FSH, a gonadotropic response was obtained with the sera for 7 weeks following withdrawal of treatment with antigens. Following a second series of treatments, sufficient anti-FSH, as well as anti-HCG, was present to mask any gonadotropic activity which the serum possessed for a 5-week period; following this the serum showed gonadotropic activity for 17 weeks. Finally, following a third series of treatment with FSH alone, no gonadotropic activity was observed for 24 weeks after the init...

Response of vitamin B6-deficient rats to non-hypophyseal gonadotropins.

SummaryThe response of Vit. B6-deficient rats to graded doses of 2 non-hypophyseal gonadotrophins, HCG and PMS, was determined and compared with that of normal immature rats. Follicular growth, cor...

I. Normal Chemical Composition and Growth

Introduction This publication relates to the chemical dynamics of skeletal growth in the normal immature male rat. Data presented are introductory to a combined chemical, radiological, and histological interpretation of skeletal growth in this animal from birth through 75 days of age.* During this period of life in the rat, individual skeletal parts as well as the skeleton as a whole undergo continuous and often disproportionate changes in growth and development.† The following studies were undertaken to correlate chemical growth and turnover of the entire skeleton with serial changes in the chemical anatomy and linear growth of long bones. Methods and Materials Male Wistarwere used throughout this study. Litters were arranged at birth to contain approximately 8 to 18male animals. All animals were weaned at 21 days of age to a standard diet (Diet 1),‡ which contained 0.76% calcium and 0.44% phosphorus by wet weight, with a calcium-phosphorus ratio of 1.7.onstant source of

Gonadotrophic hormone excretion of the pregnant monkey (Macaca mulatta).

SummaryThe urinary excretion of gonadotrophin by pregnant Macaca mulatto, was assayed at intervals from day 22 to 179 of gestation. Hypophysectomized immature female rats were injected with concentrates equivalent to 1/4 day's output of urine. Only urine excreted between days 22 and 34 of pregnancy had a gonadotrophs action, and the action was restricted to repair of the interstitial tissue. The gonadotrophic stimulus observed in normal immature rats was comparable to that from low doses of human chorionic gonadotropin during this extremely limited period of pregnancy.

Qualitative changes in urinary gonadotrophins in human pregnancy during the period of rapid increase in hormone titer.

The gonadotrophic action of the urine of pregnant women was first shown in normal immature mice and rats in which it caused enlargement of follicles and corpus luteum formation. Later, when pregnancy urine was concentrated and detoxified, it was possible to inject it into hypophysectomized immature female rats. Its only effect on the ovary of such animals was on the interstitial cells; no follicular growth or corpus luteum formation occurred. As many as 8000 units (judged by the ability to stimulate corpus luteum formation in immature normal rats) have been injected by Evans and Simpson into hypophysectomized female rats with no other gonadotrophic action than interstitial tissue stimulation. Comparable results were obtained by Claesson et al., 1948, with 6000 i.u. of highly purified HCG injected over a 4 day period and as many as 24,000 i.u. over a 16-day period. The complete gonadotrophic response to pregnancy urine observed in the normal immature animal must,