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s/ Journal of Equine Veterinary Science 34( 2014) 3939 0.5 ng/ ml, and total intra-assay coefficient ofvariation for and stallions with severe testicular dysfunction. Oligo- the assay was 2.8%. spermia was induced in the stallions using a single dose of Differences between Group TREATED and Group CON- an indenonopyridine derivative, RTI-4587-073(1)( formerly TROL were analyzed using two samples t- test. Correlations called 1- CDB-4022) which is a compound with known anti- between RI, PI, total sperm number, TNPNS, total testicular spermatogenic properties. 15]. Oligospermic stallions had volume, and FSH were tested using Pearson product- cor- resistive blood flow in the testicular artery, which was re- relation procedure. flected in high values ofRI and Pl. Control stallions( normal testicular function) had non-resistive blood flow and low 3. Results values of RI and PI. The values of RI and PI were also negatively correlated with total sperm numbers, total All stallions

The relationships between cell function and the incorporation of endogenous prolactin (PRL) by milk secretory cells (MSC) and lutein cells (LC) were examined in lactating rats which had been given graded estradiol treatment designed to dissociate progressively the activities of these two PRL target cell populations, decreasing MSC function while maintaining or augmenting LC function. MSC function was assessed by monitoring pup growth and by histological study of mammary tissue. LC function was assessed by radioimmunoassay (RIA) of circulating progesterone (P) and 20a-hydroxyprogesterone (2Ooi) and by histological evaluation of ovaries. PRL availability was assessed by RIA of serum PRL and by morphological evaluation of pituitary PRL cells. Intra cellular distributions of endogenous PRL in both target tissues were examined immunohisto chemically. The availability of PRL binding sites was also examined in MSC from rats given the highest dose of estrogen. As estrogen dosage increased, PRL secretion was maintained, or increased. Despite this, there Was progressive lactational failure, accompanied by a graded failure of PRL incorporation into MSC. These cells appeared to lose, successively, their abilities to translocate PRL to nuclei, to transport it from basal to apical regions of the cell and ultimately, with the highest dose of estro gen, to internalize or even recognize PRL, as evidenced by the absence of not only PRL, but even PRL-binding sites. In contrast, as estrogen dosage increased, titers of P and P/20o ratios remained high and LC showed progressive morphological signs of not only increased activity (hypertrophy, nucleolar enlargement) but augmented incorporation of PRL. Thus, in both MSC and LC, estrogen produced a spectrum of variations in responsiveness to PRL; this spectrum was correlated with a series of effects on PRL incorporation by these cells. These estrogenic modulations of PRL action might, therefore, involve regulating the access of this protein hormone to various intracellular control points. 394 NOLIN AND BOGDANOVE MATERIALS AND METHODS Two experiments were performed. In the first, 6 rats (CD strain, Charles River Breeding Laboratories, Inc.), each nursing her own litter of 9—li pups, were given estrogen by implantation of open-ended 5 mm sections of polyethylene tubing (innramedic PE5O, Clay Adams Co., 0.584 mm i.d. ; 0.965 mm o.d.) packed with crystalline estradiol 3,17-dipropionate (ED Lot no. 179—812, Dextran Chemicals, Inc.). A 23 gauge stylus was used to pack the tubing. Each rat was lightly anesthetized with ether on Day 4 of lactation and a short, shallow-beveled 17 gauge needle, fitted with a stylus, was used to inject 1 implant into each of 3 mammary glands. The rats were not handled again until Day 14, when they were decapitated and their ovaries, pituitaries and mammary glands were col lecned, fixed in Bouin's fluid and processed for im munohistochemical study, as described below. The second study was done to examine the possi ble dose dependency of the estrogen effects observed in the first study. Thirty pregnant rats of the same (CD) strain were used. Beginning “? vi week prior no delivery, the rats were caged individually in 23 X 20 X 14 cm clear plastic boxes with wire tops in a room with lights on 14 h/day (0500—1900 h) and an ambi cnn temperature of 23 ±1°C.Food and water were continuously available. All rats delivered on the same day (Day 1 of lactation). Immediately after delivery, pups were counted. Six rats that delivered less than 8 pups each were discarded. Some of their pups, as well as those in excess of 10/litter from the other rats, were used where necessary to equalize all litters no 10 pups. Twenty-four families were then divided into 4 groups of 6 families each. At “? “1000 h of Day 4, each mother and each litter were weighed. Mothers were non weighed again until Day 15. Litters were weighed on alternate mornings and on the morning of Day 15. Estradiol benzoate (EB, Progynon benzoane, 6CX2P96706, Corp.) was given to 18 of the mothers at “? †v1700 h on Days 4—14(11 days). Six mothers were given each dose of EB (1.0, 4.0 or 16.0 Mg)by s.c. injection in 200 p1 sesame oil (U.S.P. 0-154, Fisher Scientific Co.). The remaining 6 mothers, 3 injected with oil and 3 untreated, served as controls Since the 2 control groups did non differ, they are considered as 1 group under Results. Between 1600—1800 on Day 15, mothers were rapidly decapitated. Trunk blood was collected and refrigerated until sera could be separated an “? v1830 h. Terminal body weights were obtained by adding the weights of the trunk, head and extravasated blood. Pituinaries, mammary glands and ovaries were removed and placed in Bouin's fluid. Pups were killed by cervical dislocation. Sera collected from 2 adult male rats, killed in the same way, served as controls for the lack of stress-induced PRL release (Nolin en al., 1977). Sera were divided into 2 aliquots/sample and frozen immediately. One sen of aliquons was stored an —? 40°C until thawed for RIA of PRL, after which any residual serum was refrozen and stored further at —? 40°C. Through error, the other set was kept an —? 5°C until, 15 months later, it was assayed for progesterone (P) and 2Os-hydroxyprogesnerone (20o), together with some of the samples which had been stored an —? 40°C, using a method and antiserum described previously (Beatnie en al., 1977; Resko et al., 1974). Serum PRL radioimmunoassay was done with the NIAMDD antiserum to rat PRL (S-i), as previously reported (Nolin en al., 1977). Duncan's multiple comparison test was used for statistical evaluation of hormone concentration differences. After fixation, pituitaries, mammary glands and ovaries were dehydrated, embedded in paraffin, sectioned serially at 5 pm and stained for endogenous PRL by the immunohistochemical method described in detail previously (Nolin and Witorsch, 1976). Reagents used in the present study were: goat anti serum to monkey 7-globulin (G61-1, Antibodies, Inc.), rabbit antiserum no ran PRL (APRL: NIAMDD anti-rat PRL 53) and goat antiserum to rabbit -y globulin (AR'@C;65-159-1, Miles Laboratories). AR'yG was conjugated no peroxidase by the method of Nakane and Kawaoi (1974). Diaminobenzidine:H2 02 was used as the chromogenic substrate. Fully absorbed APRL (ABS APRL), used in the primary test for PRL specificity of the staining reac tion, was prepared by mixing 1 ml APRL (1:50) with 8 @gNIAMDD rat PRL 1-2. Material stained via APRL binding was identified as PRL only if it was tonally unstained in adjacent serial sections when ABS-APRL was used in place of APRL in an otherwise identical reaction sequence. Pituitaries were subjected to the same protocol as target tissues, providing an adjunctive nest of method specificity (see Nolin 1978a, footnote 2). Mammary glands from the ED implant group were also subjected to immunohisnochemical testing for the presence of PRL binding sites which could bind exogenous PRL (Nolin and Witorsch, 1976). For this, sections were preincubaned with exogenous PRL (NIAMDD ran PRL 1-2, in amounts ranging from 15—1500 pg/section) prior to application of APRL in the PRL-staining sequence. Maternal Weights RESULTS Data were non obtained in the first experi ment (rats with ED implants). In the second, mean maternal weight on Day 4 was 288 ±9 g. On Day 15, the mean weights for controls and the 1, 4 and 16 pg/day EB treated rats were 310 ± 11, 294± 9, 304 ±7 and 292 ±8 g, respectively. The differences among the 4 groups were not statistically significant.

Rabbit antisera to rat pituitary follicle-stimulating hormone (FSH) and to rat luteinizing hormone (LH) were used, in an immunocytochemical probe, to determine the ontogeny and distribution of immunoreactive, endogenous, intraovarian FSH and LH in immature rats. Ovaries from rats 4, 8, 12, and 21 days of age were studied. Both gonadotrophins were first immunodetectable on Day 8. In reactive primordial follicles, LH was restricted to the cytoplasm and nuclei of the surrounding follicle cells. In those follicles possessing both squamous and cuboidal follicle cells, i.e., transitional between primordial and primary, LH was found in both the cytoplasm and nuclei of both follicle cell types. In primary follicles, LH was no longer present in granulosa cells but was concentrated in germ cell cytoplasm. In contrast, in primordial follicles, FSH was restricted to the germ cell but was present in both the oocyte cytoplasm and germinal vesicle. In transitional and primary follicles, FSH remained within the oocyte cytoplasm and germinal vesicle but also became detectable within the cytoplasm and nuclei of granulosa cells. These findings raise some important new questions regarding the role(s) of the gonadotrophins in early follicular development.

In both intact and castrated male and female rats, administration of the A-ring reduced androgen, dihydrotestosterone (DHT), consistently failed to stimulate prolactin (PRL) secretion although it inhibited LH release and, in males, stimulated ventral prostate growth. In intact females, but not in the other types of rat, DHT actually suppressed PRL release. These findings do not support generalizations, based entirely on findings with testosterone, that both "androgens" and estrogens exert stimulatory actions on PRL secretion. The distinct stimulatory effects of testosterone and its esters on PRL secretion seem attributable, not to their androgenic actions per se, but to the ability of testosterone to form estrogenic metabolites. This ability does not appear to be shared by the "pure" androgen, DHT.

Ovarian follicles were examined for the presence and distribution of endogenous PRL in nor mally lactating control rats and comparable rats which had been subjected no a variety of annilacta tional, luteonropic estrogen treatments. Control ovaries contained both preantral follicles and annral follicles up to 500 @m in diameter. In healthy antral follicles, PRL was present in the cyno plasm and nuclei of granulosa cells (GC), in follicular fluid (FF) and also in the cytoplasm of the manuring dicnyane oocynes (OC), where ins lattice-like distribution resembled that of, and sug gesned an association with, yolk material. PRL was distributed similarly in OC of healthy preannral follicles, but in was absent from the surrounding GC. In anresia, both GC and OC showed disturb ances of PRL distribution (absence from GC nuclei, irregular staining of yolk lattice) or complete absence of staining for PRL. The changes in OC were seen in rats given only 4 @g estradiol benzo ate/day. Stronger estrogen treatments produced anrenicchanges in GC as well. These findings suggest at least two periods of PRL incorporation by healthy dicnyane OC: one prior no, or early in, the stage of meionic arrest, the other during preovulanory manuranional en largement. During the later period, when PRL is transported no the OC through GC cytoplasm and FF, in also enters GC nuclei. The prolonged retention of PRL within the OC and ins renewed in corporation as this cell approaches innerphase, together with recent evidence that PRL may play a role in the inhibition of meiosis in mammalian oocynes, suggest that the specific local resistance no ovulation -inducing stimuli, which is needed for both selective maintenance of the dicnyane state and fine control of preovulatory maturation, might be a function of the PRL snored within the OC itself.

This study was done to examine whether PRL, which appears in both active milk secretory cells (MSC) (1) and milk (1,2) during normal lactation, could also be detected in PRL target cells which do not transfer this hormone into an exocrine secretory product. Ovaries, adrenals, and mammary and pituitary glands were collected from actively nursing rats decapitated on day 15 post-partum. All four tissues were processed for light microscopic immunohistochemical identification of PRL. Immunoreactive PRL was again found in pituitary PRL cells and in MSC. It was also detected within both lutein and adrenal cortical cells (zona fasciculata). In all three target tissues, PRL was present in target cell cytoplasm, but, in MSC and adrenal cells, it was also found occasionally in nuclei. These findings, which seem to indicate that transfer into an exocrine secretory product cannot be the only possible explanation for the appearance of this protein hormone inside its target cells, extend the evidence suggesting that PRL may have intracellular sites of action.

Recent refinements in methodology now permit the study of endogenous peptide hormones in their individual target cells. The investigations reported here deal with the question of whether endogenous ACTH can be detected in its target cells in the highly active adrenal gland of the normally lactating rat. This question was examined with immunohistochemistry. ACTH was found in both cytoplasm and nuclei of adrenal glomerulosa cells. In cells of the fasciculata and reticularis layers of the adrenal cortex, it did not appear inside nuclei but was present in the cytoplasm and on the nuclear envelope. The distribution of ACTH was compared with and found to be different from that of PRL. PRL, confirming previous findings, was not detectable at all in glomerulosa cells and, in cells of the inner cortical zones, was present in both cytoplasm and nuclei. In neither case was hormone found in the adrenal medulla. The apparent feasibility of studying peptide regulators such as ACTH and PRL in their individual target cells may be a focal point for an acceleration of our understanding of how these peptides work.

Endocrinology can be viewed as a very old science when one considers that ablation of certain of the glands of internal secretion, in particular the gonads, was practiced very early on in our history. (One can even postulate that the bravest among some of our ancestors got hold of a saber-toothed tiger long enough to turn him into a pussycat!) It was not until whole organs, homogenates, or extracts were tested for activity that could repair a deficiency, however, that modern endocrinology began about a century ago. This sort of qualitative bioassay approach continued to be used well past the first third of the present century and involved hormones exclusively in their glands of origin, i.e., hormones in very large amounts. As bioassay sensitivities were improved, detection of a relatively wide range of concentrations became possible in the late 1940’s and 1950’s, and during this period it even became possible to detect some hormones in transit to their targets, but again, the amounts that could be detected were present at the lower concentrations that could be measured in tissues of origin, and therefore the improved methods could detect hormones in blood only if they were present in supraphysiological amounts. Then, in the late 1960’s and early 1970’s, came radioimmunoassays (RIAs) and radioreceptor assays (RRAs) that allowed the study of hormones in transit, not only in physiological, but also in subphysiological, concentrations.

Publisher Summary This chapter presents a line of evidence that for at least one anterior pituitary hormone, feedback control is not only quantitative but also qualitative. This means that the target gland—in this case, the gonad in its “back-talk” to the pituitary or brain-pituitary system—can dictate not only the amount of tropic hormone to be secreted but also the kind of tropic hormone. The chapter presents an evidence that in a rat, there is a set of changes in the biological and chemical characteristics of the FSH secreted by the pituitary that seems to bear no fixed relationship to either the degree or direction of any concomitant quantitative changes in FSH and LH secretory rates. These qualitative changes can be induced rapidly by alterations in the gonadal steroid environment to which the pituitary or brain-pituitary system is exposed. Furthermore, they can occur within a physiological range of circulating sex steroid levels. In the absence of gonadal steroids, the pituitary of the rat synthesizes, stores, and releases a type of FSH that is called “neuter-FSH.” In response to androgen replacement, both stored and circulating forms of FSH change in three ways. The pituitary begins to secrete a different hormone, “andro-FSH,” for which the index of discrimination, apparent molecular size, and capacity to survive in the circulation all are greater than they are for “neuter-FSH.” Ovarian steroid(s) appear to have an opposite effect. The index of discrimination for the FSH in female rat pituitaries is low, and the apparent molecular size of this FSH is even less than that of “neuter-FSH.” It seems reasonable to speculate that the female rat releases a relatively short-lived “gyno-FSH” into the circulation. The chapter also discusses the discovery of the types of FSH heterogeneity.

Prolactin (PRL) is a pluritrophic protein hormone that exerts multiple actions not only on different target tissues but also even within a single target cell population. Its modus operandi, however, has been elusive.

Is the entry hypothesis compatible with all the existing data about "the" second messenger for prolactin listed in Section II? All of these messengers, in some way either participate in, or modify, prolactin's actions or, in an end point-dependent manner, may actually mimic prolactin. There remains considerable uncertainty as to whether these findings reflect phenomena, some independent of and others quite dependent upon entry, on the one hand, or merely portions of a relatively large number of molecular cascades, some (but not necessarily all) begun initially at the plasmalemma and many (if not all) orchestrated toward completion by intracellular prolactin or agonist-receptor complex.

Two approaches were used to establish the intercellular distribution of fatty acid synthetase and thioesterase II in the lactating rat mammary gland. Thioesterase II is the chain-length regulatory enzyme in the biosynthesis of the medium-chain fatty acids characteristic of milk fat. Using immunohistochemical techniques, immunoreactive fatty acid synthetase was found in both mammary adipocytes and epithelial cells; in contrast, immunoreactive thioesterase II was confined to the epithelial cells. In metabolic studies, adipocytes and epithelial cells were isolated from lactating rat mammary glands after digestion with collagenase and thermolysin, and their lipogenic activity was studied using isotopically labelled acetate. Consistent with the immunohistochemical data, adipocytes synthesized exclusively long-chain fatty acids whereas epithelial cells synthesized predominantly medium-chain fatty acids. The results indicate that the capacity for synthesis of medium-chain fatty acids is a unique property of the epithelial cell component of the mammary gland.