Hormonal changes associated with ageing and induced moulting of domestic hens.

1. The effect of age on ovarian function was studied in 245-, 350-, 500-, 700- and 800-d-old Lohmann hens. The effect of three different methods for moult induction on ovarian function and corticosterone concentration was studied in 500-d-old hens. 2. No significant reductions in ovarian weight or in number of follicles before the age of 700 d were found. The ability to produce progesterone and oestradiol-17beta was unchanged up to the age of 700 d and the circadian secretion of these two steroids was identical in young (225 d) and old hens (600 d). 3. The effects of induced moulting by feed withdrawal (FW) and a high Zn (HZn) diet on body weight and ovarian function were very similar; those of a moderate Zn with low Ca (MZn/LCa) diet were smaller. 4. The first significant effect of moulting was a decrease in oestradiol-17beta plasma concentration (d 2). Plasma progesterone decreased more gradually than oestradiol-17beta, and reached a nadir on d 6 in FW- and HZn-treated hens and on d 9 in MZn/LCa-treated ones. 5. Hens treated with either FW or the MZn/LCa, but not those with the HZn diet, showed a very sharp rise in corticosterone concentration on d 2 of treatment. Thus the MZn/LCa diet was less efficient than the other treatments in induction of ovarian involution, but had a similar effect on stress induction, as indicated by increases in plasma corticosterone.

Studies in vivo and in vitro on the initiation of follicle growth in the bovine ovary.

Histological sections prepared from cortical parts of 25 bovine ovaries were used to study initiation of follicle growth in vivo. Small follicles were measured and characterized. Initiation of follicle growth consisted of two distinct consecutive phases. The first phase was characterized by transformation of granulosa cells from a flattened to a cuboidal shape and by their proliferation. In the second phase an increase in the number of granulosa cells was accompanied by a rapid increase in the size of the oocyte. Oocytes commenced growth when there were at least 40 granulosa cells in the largest cross-section (fourth generation of follicle cells). The oocyte diameter increased from 29.74 +/- 0.30 microns (mean +/- SEM) in primordial follicles to 92.90 +/- 4.50 microns in small antral follicles. The zona pellucida first appeared as an island of periodic acid-Schiff positive material in small preantral follicles, but formed a complete ring around the oocyte when the late preantral stage was reached. Organ culture of ovarian cortical explants was used to study initiation of follicle growth in vitro. Within 2 days of culture most of the primordial follicles entered the growth phase: granulosa cells changed from a flattened to a cuboidal shape and entered S-phase as demonstrated by autoradiography after [3H]thymidine incorporation. On day 2, 48.6% of follicles were labelled compared with 3% on day 0. Follicle growth started in the absence of gonadotrophins, in the serum-free medium, confirming the notion that gonadotrophins are not essential for this process. The culture system used here will be helpful in the study of the involvement of putative factor(s) in the initiation of follicle growth in large domestic animals.

Differential expression pattern of inhibin alpha and betaA subunits in the ovaries of postnatal and prepubertal lambs.

The aim of the present study was to characterize gene and protein expression of follistatin, inhibin alpha (alpha) and inhibin betaA (betaA) subunits in the ovaries of postnatal (3-week-old) and prepubertal (14 and 20-25-week-old) lambs. Northern blot analysis revealed the presence of two alpha and two betaA mRNAs. In postnatal ovary the a 1.2-kb transcript was abundant, its amount gradually falling, while the 2.0-kb mRNA increased and became a major band at 20-25 weeks. Both betaA mRNAs, 4.5 kb and 6.0-7.5 kb, were weakly expressed in postnatal ovary, but whereas the 4.5-kb mRNA expression remained at a low level, that of the 6.0-7.5 kb mRNA increased about five fold in prepubertal ovary. The ratio of total alpha mRNAs to the dominant betaA form (6.0-7.5 kb) varied from 1.27 at 3 weeks to 0.33 at 20-25 weeks of age. One major follistatin mRNA of 2.5-3.6 kb was recognized and was constitutively expressed during ovarian growth. Several molecular-mass forms of alpha and betaA subunits with different compositions were seen in prepubertal compared with postnatal ovaries, the latter exhibiting more active follicular growth. In summary, ovine ovaries undergo distinct changes early in life, both morphological and functional, and show a changing pattern of inhibin subunit expression.

Changes during puberty in chromatin condensation, morphology and fertilizing ability of epididymal spermatozoa of the golden hamster.

Male golden hamster sperm acquire complete fertilizing ability at about 48 days of age. In this study hamsters, 27-130 days of age were killed and their male reproductive tracts examined. Sperm were found in the caudae epididymides from 37 days onward. None of the sperm from animals younger than 41 days were capable of fertilizing ova when placed in the uteri of superovulated females. Using flow cytometry of acridine-orange-stained cells, the chromatin condensation in cauda epididymal sperm was investigated. It was seen that DNA from sperm from the younger animals (under 40 days of age) was less tightly bound to protamine than that obtained from mature animals. In summary, the earliest sperm produced by pubertal hamsters were immature with regard to chromatin condensation, morphology, motility, and ability to fertilize ova, and they developed mature characteristics in the period between 40-48 days of age.

Investigation of epididymal sperm maturation in the golden hamster.

During passage of hamster spermatozoa through the epididymis their maturation is shown to involve changes in the sperm head, midpiece (mitochondria) and tail. The sum of these changes results in a dramatic increase in the fertilizing potential of the spermatozoa. When comparable numbers of spermatozoa from the caput or corpus epididymis were injected into one uterine horn of mature females, following ovulation induction, and spermatozoa from the cauda epididymis were injected into the contralateral horn, no fertilization was observed with caput epididymal spermatozoa, 1.7% of oocytes were fertilized by corpus epididymal spermatozoa, whereas 79.5% fertilization was obtained with cauda epididymal spermatozoa. Total sperm numbers increased from caput to corpus to cauda [28.3 +/- 12.2, 40.6 +/- 20.8, 144 [corrected] +/- 62 million, respectively]. The percentage of progressively motile spermatozoa increased from 27.9 +/- 6.4 to 33.8 +/- 4.8 to 70 +/- 10.7 during this passage. Viability, measured by exclusion of the dye, propidium iodide, was significantly less in spermatozoa from the cauda than from the proximal or mid-caput epididymis. The percentage of the live cells that were stained intensely by rhodamine-123 (a measure of mitochondrial membrane potential) increased during epididymal passage from 22.8 +/- 7.8% in the proximal caput epididymis to 57.2 +/- 16.5% in the cauda epididymis. Staining with acridine orange (a measure of DNA packaging in the sperm head) indicated an increase in chromatin condensation in cauda epididymal spermatozoa, when compared to those obtained from the caput or corpus.

Chromatin condensation in hamster sperm: a flow cytometric investigation.

In this study we have used acridine orange staining, as described by Evenson (1990), to follow changes in DNA packaging as they occur in hamster spermatozoa which have left the testis and are undergoing maturation in the epididymis. Measurement of the green and red fluorescent intensities of hamster sperm nuclei by flow cytometry demonstrated a decrease in acridine orange binding to DNA as sperm made their way from proximal corpus epididymis to the vas deferens. Using sperm from the cauda epididymis of the mature hamster as the standard, a method was developed for estimating the % of cells in a given sample that have matured with regard to DNA packaging. Staining with bromobimane was used to determine the extent of sulfhydryl oxidation in the nuclei. It was seen that sulfhydryl oxidation occurred mainly in the cauda epididymis whereas another process in chromatin condensation occurred earlier, during sperm passage through the caput epididymis. This earlier process could be mimicked by incubating sperm nuclei with alkaline phosphatase, suggesting that it consists of removal of phosphate in protamine.

Protection against autoimmune disease by bacterial agents. II. PPD and pertussis toxin as proteins active in protecting mice against experimental autoimmune encephalomyelitis.

Bordetella pertussis and Mycobacterium tuberculosis, routinely used to promote the development of autoimmune disease, were recently reported to also be effective in inducing protection against an autoimmune disease. Thus, we previously demonstrated that SJL/J and (SJL/J x BALB/c)F1 mice that are genetically susceptible to experimental autoimmune encephalomyelitis (EAE) become highly refractory to the induction of the disease following their exposure to B. pertussis and M. tuberculosis. In the present study, the pertussis toxin (PT) from B. pertussis and the purified protein derivative (PPD) of M. tuberculosis, were found to be sufficient to fully protect against EAE and thus may be the major bacterial components responsible for conferring protection. The 65-kDa heat-shock protein played only a marginal role in the protection against EAE induced by these bacteria. Both PT and PPD were protective when given before, but not after, the encephalitogenic challenge, and minute amounts (5-50 ng) emulsified in oil were sufficient to confer long-lasting resistance to EAE. The effect of PT or PPD on EAE differed from that of mitogens or bacterial superantigens, suggesting that their protection ability was not attributable merely to mitogenic or superantigenic properties. The mechanism of protection is not yet clear. Preliminary studies revealed a complex mechanism of protection whereby PPD and PT may operate differently. Thus, only PPD-induced, but not PT-induced, protection was transferrable by CD4+ T lymphocytes bearing an alpha beta T cell antigen receptor. Neither PT nor PPD had a protective effect on EAE mediated by preformed pathogenic T lymphocytes and it is most likely that they exert their protection by affecting the development of such T lymphocytes. How bacteria such as B. pertussis and M. tuberculosis can either enhance the development of an autoimmune disease or protect against the disease is not yet clear. However, identifying PT and PPD as the bacterial components active in protection may allow a better understanding of the modulatory effects of bacteria and point to the potential use of such bacterial products in immunomodulation of autoimmune diseases.

Staphylococcal enterotoxin B as a potent suppressant of T lymphocytes: trace levels suppress T lymphocyte proliferative responses.

Staphylococcal enterotoxins have long been known to be powerful stimulators of T lymphocytes in mouse and man. In a previous study we showed that high concentrations of staphylococcal enterotoxin serotype B (SEB) failed to stimulate strong proliferative responses by Lewis rat T lymphocytes. Moreover, concentrations of SEB (10-50 micrograms/ml) that stimulated optimal mouse T lymphocyte proliferative responses suppressed a mitogen- or antigen-induced rat T lymphocytes proliferative responses. The present study shows that SEB at low concentrations (as low as 10(-3)-10(-4) micrograms/ml) and often also trace levels (about 10(-6)-10(-7) micrograms/ml) suppresses both rat and mouse T lymphocytes proliferative responses to mitogen or antigen. Furthermore, under different circumstances, SEB may have conflicting effects on the same T cells. While high concentrations (1-50 micrograms/ml) of SEB stimulate certain mouse T cell clones, low concentrations or trace levels have a potent suppressive effect on the same clones. The results indicate that the in vitro conflicting effects of SEB on the same T cells are concentration dependent and may reflect its in vivo effects on SEB-reactive T lymphocytes. The suppression of the mitogen- or antigen-induced stimulation of T cell clones by SEB was direct and did not require the agency of suppressor cells. Furthermore, the suppression by low amounts of SEB was not major histocompatibility complex restricted and affected a large proportion of both rat and mouse T lymphocyte subpopulation, regardless of their antigenic specificity. The concomitant suppressogenic and stimulatory characteristics of SEB support the conclusion that, under different conditions, SEB can be considered a "super-suppressogen" as well as a "super-antigen". Overall, the results suggest that SEB, and possibly other bacterial toxins, could be useful in immunomodulation of specific T cell responses.

Reversal of autoimmune encephalomyelitis by membranes presenting myelin basic protein-associated class II MHC molecule as an approach to immunotherapy of organ-specific autoimmune diseases.

Experimental autoimmune encephalomyelitis (EAE), a well-accepted experimental model for multiple sclerosis in humans, is a paralytic disease mediated by CD4+ T cells specific for myelin basic protein (MBP). Several approaches to immune-specific therapy of EAE as a model for other organ-specific autoimmune diseases have previously been reported. We now show that macrophages (M phi) or B cells, as antigen-presenting cells, when pulsed with MBP and intraperitoneally (but not intravenously) inoculated after the encephalitogenic challenge, are highly effective in blocking the development of EAE. Moreover, M phi pulsed with an organ tissue homogenate, mouse spinal cord homogenate, can also present the relevant target antigen, MBP, and are as effective as MBP-pulsed M phi in blocking the development of EAE. This capacity of the M phi to identify and present the relevant target antigen indicates that this approach is also applicable to organ-specific autoimmune diseases other than EAE, regardless of how much is known about their etiological agent or specific target antigen. Nonviable glutaraldehyde-fixed MBP-pulsed M phi or membranes derived from MBP-pulsed M phi retain their capacity to block the development of EAE.