Delayed anovulatory syndrome induced by estradiol in female C57BL/6J mice: age-like neuroendocrine, but not ovarian, impairments

These studies describe induction of a delayed anovulatory syndrome (DAS) by estradiol (E2) in female C57BL/6J mice. Six days after birth, female mice were injected s.c. with 0.1 micrograms estradiol benzoate or oil. Over 90% of the oil-injected controls exhibited estrous cycles from 2 to 9 mo of age. In contrast, 60% of the E2-injected mice exhibited estrous cycles at 2 mo of age but were acyclic by 9 mo; these mice were considered to have exhibited a DAS, and had longer cycles than controls. At 12 mo, ovarian impairments were assessed by examining 1) ovulation after s.c. injection of 5 IU human chorionic gonadotropin (hCG), and 2) estrous cycles after grafting into young (3-mo-old) hosts. Simultaneously, neuroendocrine impairments were assessed by examining 1) the surge of luteinizing hormone (LH) induced by E2 implants after ovariectomy, and 2) estrous cycles after receiving ovarian grafts from 3-mo-old mice. Ovaries from DAS and control mice ovulated equally in response to hCG. Ovaries from DAS mice grafted into young ovariectomized hosts supported 30% more cycles, of shorter period, compared with ovaries from control donors. However, the E2-induced LH surge was 50% smaller in DAS mice than in controls. Ovariectomized DAS hosts with ovarian grafts from young mice supported 70% fewer estrous cycles, of longer period, compared with ovariectomized control hosts with young grafts. We conclude that the E2-induced DAS in female mice is not due to ovarian impairments, but seems to result from neuroendocrine impairments.     

Age of ovary determines remaining life expectancy in old ovariectomized mice

We investigated the capacity of young ovaries, transplanted into old ovariectomized CBA mice, to improve remaining life expectancy of the hosts. Donor females were sexually mature 2-month-olds; recipients were prepubertally ovariectomized at 3 weeks and received transplants at 5, 8 or 11 months of age. Relative to ovariectomized control females, life expectancy at 11 months was increased by 60% in 11-month recipient females and by 40% relative to intact control females. Only 20% of the 11-month transplant females died in the 300-day period following ovarian transplantation, whereas nearly 65% of the ovariectomized control females died during this same period. The 11-month-old recipient females resumed oestrus and continued to cycle up to several months beyond the age of control female reproductive senescence. Across the three recipient age groups, transplantation of young ovaries increased life expectancy in proportion to the relative youth of the ovary. Our results relate to recent findings on the gonadal input upon aging in Caenorhabditis elegans and may suggest how the mammalian gonad, including that of humans, could regulate aging and determine longevity.     

Mechanism of the development of permanent estrus in rats after transplantation of the ovaries into a low temperature medium

Autotransplantation of the ovaries to the ears of adult rats induces permanent estrus following 5-7 days. Autotransplantation of the ovaries beneath the renal capsules makes the sexual cycle return to normal after the same period. Autotransplantation of the ovaries to the ears of infantile rats brings about pubertas precox followed by normal sexual cycle. Permanent estrus ensues only after 4 months. Transplantation of the ovaries from infantile rats to adult ones and vice versa has shown that age-related differences in the alterations seen in the sexual cycle in response to gonadal transplantation to the ears are caused by age-related differences in the recipients, precisely by those in the cyclic center.     

Ultrastructural characteristics of the vaginal epithelium of neonatally estrogenized mice in response to subsequent estrogen treatment.

Adult mice which had received 10 daily injections of 20 microng estradiol beginning with the day of birth were in a "persistent-estrous" state, showing ovary-independent proliferation and cornification of the vaginal epithelium. Ultrastructural changes of the vaginal epithelium in neonatally estrogenized mice was examined after a single postpuberal injection of 10 microng estradiol and compared with those seen in normal mice to estrogen. In ovariectomized normal mice, the basal cells were round. The nucleus was polygonal and contained peripheral condensed chromatin. After estradiol treatment, the basal cells became columnar. The nucleus was round to oval, containing dispersed chromatin. In neonatally estrogenized ovariectomized mice, the basal layer of vaginal epithelium consisted of round cells with polygonal nuclei, much as in normal ovariectomized mice. The nucleus occupied a large area of the cytoplasm and contained prominent nucleoli. Intercellular spaces were moderately distended. Late estradiol treatment resulted in distended intercellular spaces and in the appearance of the other cell type along with round cells in the basal layers: the columnar cells containing an oval nucleus with dispersed chromatin, resembled the basal cells in normal ovariectomized mice receiving postpuberal estrogen injection. The intercellular spaces between the columnar cells were narrow compared with those between round cells. However, the nuclei of round cells still had prominent nucleoli and peripheral condensed chromatin regardless of subsequent estrogen treatment. This fact suggests that these nuclei do not respond to estrogen. These results clearly show that the vaginal epithelium of neonatally estrogenized mice with ovary-independent persistent cornification consists of a mixed population of cells.     

Estradiol-induced adult anovulatory syndrome in female C57BL/6J mice: age-like neuroendocrine, but not ovarian, impairments

Long-term effects of elevated plasma estradiol (E2) on ovarian and neuroendocrine functions were examined in 4-month-old cycling female C57BL/6J mice injected s.c. with 0.2 or 0.05 mg estradiol valerate (EV), or oil. Within 7 days, EV-injected mice became permanently acyclic, exhibiting the persistent vaginal cornification (PVC) characteristic of reproductive senescence in rodents. Four months after injection, ovaries from EV-injected mice exhibited no corpora lutea, but ovulated in response to an injection of human chorionic gonadotropin (hCG) (as do older, spontaneously PVC mice). When grafted into young mice, ovaries from EV-injected mice supported as many estrous cycles as ovaries from oil-injected controls. EV did not alter the suppression of luteinizing hormone (LH) by E2, LH response to injected LH releasing hormone (LHRH), or plasma prolactin (Prl). However, EV-injected mice exhibited impairments in LH regulation similar to those seen in old, acyclic mice. Plasma LH 30 days after ovariectomy was 40% lower, and E2-induced LH surges were 60% lower, in EV-injected mice versus controls. Furthermore, EV-injected mice were unable to support estrous cycles given young ovarian grafts, in contrast to controls. Effects of sustained but physiological levels (15-20 pg/ml) of plasma E2, were examined in intact cycling mice given sham or E2 implants. Six weeks after implantation, the implants were removed; only 50% of the E2-implanted mice subsequently exhibited estrous cycles, compared with 100% of sham-implanted controls. Furthermore, those E2-implanted mice which did cycle had fewer cycles than controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Orthotopic ovarian transplantations in young and aged C57BL/6J mice

Orthotopic ovarian transplantations were done between young (6-wk-old) and aged (17-mo-old) C57BL/6J mice. The percentages of mice mating following surgery from the four possible ovarian transfer combinations were as follows: young into young, 83%; young into aged, 46%; aged into young, 83%; and aged into aged, 36%. The percentages of these mice that were pregnant 10 days following the presence of a vaginal sperm plug were as follows: young into young, 58%; young into aged, 9%; aged into young, 50%; and aged into aged, 0%. Some of the fetuses derived from matings of the above mice were dissociated and their cells prepared for chromosomal smears. No evidence of aneuploidy or mosaicism was found in fetuses derived from ovaries of young or aged mice. Aged ovaries, transferred to either young or aged recipients, were found to have fewer developing follicles and lower weight, which was most apparent in recipients that failed to mate or to get pregnant. Concentrations of luteinizing hormone, follicle-stimulating hormone (FSH), and prolactin in plasma from each of the pregnant recipients were analyzed by radioimmunoassay. The only statistical differences found between the transfer groups occurred in FSH concentrations. Plasma FSH was markedly elevated (P less than 0.005) in young recipients with ovaries transplanted from aged donors, in comparison to young recipients with ovaries from young donors. These data indicate that the aging ovary and uterus play a secondary role in reproductive failure and that the aging hypothalamic-hypophyseal complex is primarily responsible for the loss of fecundity in older female C57BL/6J mice.     

Endogenous oocyte antigens are required for rapid induction and progression of autoimmune ovarian disease following day-3 thymectomy

Female (C57BL/6xA/J)F(1) mice undergoing thymectomy on day 3 after birth (d3tx) developed autoimmune ovarian disease (AOD) and autoimmune disease of the lacrimal gland. As both were prevented by normal adult CD25(+) T cells, regulatory T cell depletion is responsible for d3tx diseases. AOD began as oophoritis at 3 wk. By 4 wk, AOD progressed to ovarian atrophy with autoantibody response against multiple oocyte Ag of early ontogeny. The requirement for immunogenic endogenous ovarian Ag was investigated in d3tx female mice, d3tx male mice, and d3tx neonatally ovariectomized (OX) females. At 8 wk, all mice had comparable lacrimalitis but only those with endogenous ovaries developed AOD in ovarian grafts. The duration of Ag exposure required to initiate AOD was evaluated in d3tx mice OX at 2, 3, or 4 wk and engrafted with an ovary at 4, 5, or 6 wk, respectively. The mice OX at 2 wk did not have oophoritis whereas approximately 80% of mice OX at 3 or 4 wk had maximal AOD, thus Ag stimulus for 2.5 wk following d3tx is sufficient. AOD progression requires additional endogenous Ag stimulation from the ovarian graft. In mice OX at 3 wk, ovaries engrafted at 5 wk had more severe oophoritis than ovaries engrafted at 6 or 12 wk; moreover, only mice engrafted at 5 wk developed ovarian atrophy and oocyte autoantibodies. Similar results were obtained in mice OX at 4 wk. Thus endogenous tissue Ag are critical in autoimmune disease induction and progression that occur spontaneously upon regulatory T cell depletion.     

Comparative fertility of normal and repeat-breeding cows as embryo recipients

Morphologically normal embryos were transferred surgically into uteri of normal and repeat-breeder cows at seven days post-estrus to compare embryo survival rates in the two kinds of cows. All cows were less than ten years of age and had no abnormal genital discharges, cystic ovarian follicles, or anatomical abnormalities of the reproductive tract. Normal cows had not been inseminated after last calving. Repeat-breeders had at least four infertile services within the past six months (average of 6.2 services after calving). To test fertility of repeat-breeders at synchronized estrus, 22 anatomically-normal repeat-breeders were treated by intramuscular (i.m.) injection with prostaglandin F(2)alpha (PGF(2)alpha) on day 11 of an estrous cycle (estrus = day 0) and inseminated at induced estrus; 11 cows (50%) had a normal fetus at necropsy on day 60. Twenty-three repeat-breeders and 23 normal cows were assigned as embryo recipients and treated i.m. with PGF(2)alpha to synchronize estrus. All embryo donors were normal cows. Donors were treated with FSH and PGF(2)alpha and inseminated at estrus. On day 7 after estrus, embryos were recovered nonsurgically from donors and one embryo was transferred through a flank incision to the anterior end of the uterine horn adjacent to the corpus luteum of each recipient. Recipients that did not return to estrus were necropsied at day 60. Of 28 normal and 23 repeat-breeder recipients, 23 normal cows (82%) and 16 repeat breeders (70%) were pregnant at day 60 (P=0.235). Thus, at seven days post-estrus, the maternal environment of most of these repeat-breeders was satisfactory for maintaining pregnancy.     

Ovarian activity and uterus organometry in delayed puberty gilts

About 30% of the total number of gilts selected for reproduction at the large breeding farm units in Vojvodina (Republic of Serbia) are culled due to prolonged pre-insemination anoestrus (estrus not detected until 8 mo of age). The aim of this study was to provide the answer to the following question: do the culling gilts reach cyclic ovarian activity at all? One hundred seventy five culled gilts in which external estrus manifestations were not detected by 8 mo of age were sacrificed and their reproductive organs were examined for determination of sexual maturity (ovaries exhibiting pre-ovulatory follicles 8 to 11 mm in diameter, corpora hemorrhagica, corpora lutea and corpora albicantia). Uterine weights and horn length were also determined. Functional ovaries were observed in 107 (61.1%) examined gilts, with 62 animals having one and 45 having two puberty ovarian cycles (57.9% and 42.1%, respectively). Pathomorphological changes which could result in prolonged pre-insemination anoestrus were not observed on the reproductive organs of sexually mature gilts. Our results indicate that most of the culling gilts have reached cyclic ovarian activity. The main reason for culling due to the absence of external estrus manifestations in sexually mature gilts could be inadequate estrus detection technology.     

Occurrence of permanent changes in vaginal and uterine epithelia in mice treated neonatally with progestin, estrogen and aromatizable or non-aromatizable androgens.

Female mice of the C57 Black/Tw strain were injected daily with 100 microng testosterone, 50 microng testosterone propionate (TP), 100 microng 5 alpha-dihydrotestosterone (DHT) or 50 microng 5 alpha-dihydrotestosterone propionate (DHTP), for 10 days from the day of birth. Two other groups of female mice were given neonatal injections with 20 microng estradiol-17 beta and 100 microng progesterone for 10 days, respectively. All mice were ovariectomized at 60 days of age and killed at 90 days. In 100% of neonatally estrogenized or androgenized, ovariectomized mice, the cranial part of the vagina was lined with stratified epithelium with either cornification or parakeratosis or mucification. Stratification only or stratification with superficial squamous metaplasia or cornification took place in the uterine epithelia of 18% of the TP-treated, 75% of the DHT-treated and 50% of the DHTP-treated, ovariectomized mice. In contrast, neonatally estrogenized, ovariectomized mice did not show the estrogen-independent, persistent uterine changes. Neonatal progesterone treatment failed to induce the permanent changes in the vaginal and uterine epithelia.     

Effects of x-ray irradiation on the subsequent gonadotropin secretion in normal and neonatally estrogenized female rats.

Female rats were irradiated with 190R of X-rays at 10 days of age and sacrificed 4, 7 or 12 months later. Their ovaries were histologically examined and serum levels and pituitary contents of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were determined by radioimmunoassay. Both serum levels and pituitary contents of LH and FSH rose significantly 4 and 7 months after irradiation, although the ovaries were markedly reduced in weight. On the contrary, 12 months after irradiation, the ovaries increased in weight and consisted mostly of polyhedral, hyperplastic interstitial cell masses, and both LH and FSH in the serum and pituitary were reduced to normal levels. These characteristic changes in the ovarian weight and histological appearance could not be observed in the similarly irradiated animals which were received daily injections of estrone for the first 30 days of postnatal life, i.e., daily injections of 50 mug for the first 10 days, 100 mug for the middle 10 days and 200 mug for the last 10 days. Serum LH levels of the estrogenized irradiated rats at 7 or 12 months of age did not elevate although those of FSH were significantly higher than the non-irradiated intact levels. From these results, a rise in the blood levels of LH and the FSH may be attributed to the increase in weight and the histological changes in the ovaries of the irradiated female rats, and the elevation of only FSh level may not result in the abnormal growth of the irradiated ovaries.     

Prolongation and cessation of estrous cycles in aging C57BL/6J mice are differentially regulated events

The relative contributions of ovarian failure and hypothalamic-pituitary dysfunction to the prolongation and cessation of estrous cycles were assessed by measuring the ability of acutely ovariectomized (OVX) middle-aged (12 mo) mice to cycle after receiving grafts (under the renal capsule) of ovaries from young (2 mo) mice. The potentially disruptive effect of the acyclic state on the cycling response to grafted, young ovaries was avoided restricting grafting to middle-aged hosts that were still cycling. The effect of chronic exposure to ovarian secretions before the cessation of cyclicity on age-related hypothalamic-pituitary dysfunction was also assessed. The cycling ability of long-term OVX middle-aged mice (i.e., OVX at 3 mo) bearing grafts of young ovaries was compared to that of age-matched acutely OVX controls. Grafted young ovaries extended the cycling lifespan of acutely OVX middle-aged hosts by 60%. The length of this extended cycling lifespan, however, was only 80% of that achieved by young hosts bearing grafts of young ovaries. Young ovaries in middle-aged mice markedly lowered the incidence of long cycles (greater than 5 days), shifting the modal cycle length to 5 days. However, young ovaries in middle-aged mice failed to increase the incidence of 4-day cycles, the modal cycle of young controls. Middle-aged ovaries grafted into young hosts lengthened their cycles and shortened their cycling lifespan to middle-aged values. Long-term ovariectomy failed to increase the cycling lifespan of middle-aged hosts bearing grafts of young ovaries beyond that achieved in acutely OVX mice. Long-term ovariectomy did shorten the modal cycle length of middle-aged mice to 4 days, although the duration of 4-day cycling was only one-third (2 mo) that of young controls. These results indicate that the relative contributions of ovarian and neuroendocrine factors to three major events of reproductive aging vary with each event. Whereas the hypothalamic-pituitary unit appears to play an important role in the initial shift from 4- to 5-day cycles, the aging ovary plays the major role in the subsequent shift to longer cycles and in the ultimate cessation of cyclicity. Although chronic exposure to ovarian secretions during the period of cyclicity does not play a major role in the cessation of cyclicity, it appears to contribute to the hypothalamic-pituitary changes responsible for the initial shift from 4- to 5-day cycles.     

Autograft of vitrified mouse ovaries using ethylene glycol as cryoprotectant.

Ovaries from 8 to 10-week-old N MRI mice were vitrified using RPMI solution containing 30% (W/V) ficoll 70, 0.5 M sucrose, 10.7% (V/V) acetamide and 40% (V/V) ethylene glycol (EGFS40%), and were stored in liquid nitrogen. After warming at 25 degrees C in 1 M sucrose solution and equilibration with RPMI medium, the vitrified and fresh ovarian tissues were autografted intraperitoneally. After one and two estrus cycles the animals were sacrificed and the recovered grafts were examined histologically. Five days after transplantation the vitrified ovaries they were invaded by fat and fibrous cells and the large preantral and antral follicles were degenerated. At 11 days postgrafting the stroma was devoid of necrotic cells and contained normal primordial and primary follicles, suggesting that the vitrification is a simple, useful and efficient procedure for cryopreservation of murine ovarian tissues.     

Spermatogenesis in testis xenografts grafted from pre-pubertal Holstein bulls is re-established by stem cell or early spermatogonia

Xenografting of testis explants into recipient mice has resulted in successful restoration of spermatogenesis in several species. Most studies have utilized neonatal donor tissue, although a few have used prepubertal testes. In Holstein bulls, prepubertal development of the testis occurs between 16 and 32 weeks of age. The purpose of the present study was to determine the optimal age during prepubertal development of Holstein bulls for testis grafting. Explants of testis tissue from Holstein bulls between 12 and 32 weeks of age (2 bulls/age; 6 ages) were subcutaneously grafted into castrated or intact immunocompromised mice (n=8/age), then recovered after 75 and 173 days (n=4 mice/grafting period) and evaluated histologically for spermatogenic progression. Seminiferous tubules were assigned a score based on the most advanced type of germ cell present within the tubule and the average for all tubules scored (n=25) within an explant was calculated. Scores for all explants per mouse (n=6) were averaged to give a single spermatogenic progression score per mouse. No difference in spermatogenic progression of grafts between intact and castrated recipients was observed. Spermatocytes were observed in testis grafts from bulls of all ages 75 days post-grafting. At 173 days, the spermatogenic progression score for explants derived from 20 weeks bulls was greater than all ages except 12 weeks donors (p<0.05), with 8% of tubules containing spermatids. Donor material from bulls older than 20 weeks had lesser spermatogenic progression scores largely attributed to the greater number of atrophic tubules in grafts from older donors. Grafts from 28 and 32 weeks donors showed signs of degeneration by 75 days post-grafting, with 30 and 55% atrophic tubules, respectively, and lesser spermatogenic efficiency scores. By 173 days post-grafting, 72% of tubules in explants from 32 weeks donors were atrophic. The results of the present study suggest that the early stages of prepubertal development are optimal for testis grafting while advanced spermatogenesis in the donor tissue prior to grafting had a negative effect on graft development. Spermatogenesis within the grafts apparently needs to be re-established by spermatogonial stem cells or early spermatogonia.     

Chemical properties of bovine cervical mucus during normal estrus and estrus induced by progesterone and/or PGF2alpha

Ninety-two Friesian cows were used to determine the chemical properties of cervical mucus during normal estrus and estrus induced by progesterone (P4)-releasing intravaginal devices (PRID) and/or prostaglandin F2alpha. The animals were assigned to 4 groups (no treatment, a PRID for 12 days plus injection of 1000 IU PMSG at the removal of PRID, a double i.m. injection of PGF2alpha 11 days apart, or PRID for 7 days plus an im injection of PGF2alpha 24 h before the removal of PRID). A number of cows with normal estrus exhibited three consecutive estrous cycles after delivery. Cows that had not shown estrus for 3 months after delivery had their ovaries palpated twice at 10-day intervals, to determine their ovarian activity. Then PRID and/or PGF2alpha was administered in cows that had a palpable corpus luteum in one of the two palpations (cyclic cows). A double artificial insemination (AI) was performed to the cows of the three induced-estrus groups, while the cows with normal estrus received only one AI. Cervical mucus samples were collected from all cows 5 to 30 min before the first AI. Additionally, samples of cervical mucus were collected from 20 cows during their first estrus after the induced one. The results are summarized as follows: 1) The biochemical properties of cervical mucus in the first three estrus periods after delivery were similar. 2) These properties were similar both in normal estrus after delivery and in the first estrus after an induced one. 3) Glucose and fructose concentrations for normal estrus were similar to those for induced estrus groups. 4) Total protein and cholesterol concentrations were significantly lower (P < 0.001) in normal than in induced estrus, while no difference was found among the induced estrus groups. 5) Pregnancy rates of the cows did not differ significantly among the normal and the induced-estrus groups. 6) The percentages of cows in the induced-estrus groups that produced cervical mucus with total protein and cholesterol concentrations similar to those for the normal estrus groups was very low.     

Increases in the basal secretion rate of follicle-stimulating hormone (FSH) accompany age-associated changes in serum FSH levels on estrus

This laboratory has recently reported that by 5-6 months of age, alterations in the secretion and production of follicle-stimulating hormone (FSH) occur in virgin female rats which precedes the age-related disruption of estrous cycles and attenuation of preovulatory gonadotropin surges. Specifically, circulating immunoreactive FSH levels are higher on estrus in rats 5 months and older compared to levels measured in 2- to 3-month-old rats. Therefore, the present study was conducted to explore a possible mechanism for this age-related increase in FSH levels. At 1400 hr on proestrus, estrus and diestrus-1, groups (n = 6-12 rats/group) of 3- and 7-month-old, cyclic rats were decapitated, trunk blood was collected, and anterior pituitary glands were bisected and placed in incubation flasks containing 1 ml media (medium 199). Following a 30-min preincubation period, hemipituitary fragments were incubated for an additional 2 hr. Media and serum FSH levels were quantified by RIA. Levels of FSH were twofold higher in the serum of 7-month-old rats than 3-month-old rats on estrus. Similarly, the basal secretion rate (BSR) of FSH (expressed as ng FSH/ml/2 hr) was significantly (P less than 0.05) higher from incubated hemipituitary fragments of 7-month-old estrous rats than from fragments obtained from younger estrous rats (7 month: 1637 ng/ml/2 hr vs 3 months: 1253 ng/ml/2 hr). Neither the serum FSH levels nor the BSR of FSH differed between age groups on proestrus or diestrus-1. These results show that age-associated increases in circulating FSH levels on estrus may be attributed to an enhanced basal secretion of FSH from the pituitary gland.     

Ovarian reproductive function after exposure to diethylstilbestrol in neonatal life

Inbred and random-bred NMRI mice were treated with diethylstilbestrol (DES, 5 micrograms per day) or vehicle (olive oil) on Days 1-5 after birth. At the age of 8 wk, females were treated with saline or eCG and hCG to induce ovulation. Ova never occurred in the ampulla of the uterine tube of saline-treated, DES-treated females when these mice were not mated. After gonadotropin treatment, ova were found in the ampulla of all olive oil-treated females and in approximately 80% of DES-treated females. The number of ovulated ova was similar in both groups. Twenty percent of gonadotropin-treated, DES-treated females had ova in the ampulla and a vaginal plug after being caged with males but none became pregnant. Ovaries from inbred control or DES-treated females were grafted to the ovarian bursa of control or DES-treated ovariectomized hosts. DES-treated hosts, carrying control or DES-exposed ovaries, never became pregnant. Control females, with control ovaries or DES-exposed ovaries, became pregnant; pregnancy rate and litter size were similar for control mice regardless of whether they were supporting DES-exposed or control ovaries. Oocytes from ovaries exposed neonatally to DES can thus give rise to apparently normal offspring. The results also indicate DES-induced nonovarian disturbances, e.g. tubal and/or endometrial function, both of which are important for fertility. In the grafting experiments, a high mortality rate was found in inbred DES-exposed females caged with males. All deaths were associated with vaginal concrements (vaginal stones) and intestinal complications.     

Culture and long-distance shipment of swine embryos 1

Embryo culture techniques were used to maintain the viability of swine embryos during shipment from the United States to England. Embryos recovered surgically from Chester White and Hampshire sows and gilts were shipped to England, then transferred to Large White gilts. Estrus was synchronized between donors and recipients by including allyl trenbolone in the daily feed for 18 and 17 consecutive days, respectively. Daily dose of allyl trenbolone was 15 mg for recipients and either 15 or 20 mg for donors. Donors were mated or artifically inseminated one to three times during estrus. During shipment, embryos were cultured in groups of six to ten in polystyrene tubes (12 × 75 mm) containing 2 ml of a modified Krebs-Ringer bicarbonate medium. The gas phase was 90% nitrogen, 5% oxygen, and 5% carbon dioxide. Culture temperature was maintained at 35 C with a temperature-controlled box, and shipment was by commerical airline. Embryos were inserted into recipient uteri 20.5 to 27 hr after recovery. In total, 227 eggs were transferred to 12 recipients, resulting in the birth of seven litters totaling 58 pigs.     

Antigen recognition and IL-2 receptor gene expression as evidence against clonal deletion in mice with neonatally induced transplantation tolerance.

Neonatal transplantation tolerance was induced in B10.A mice by the injection of spleen and bone marrow cells from semiallogeneic [C57BL/10(B10) x B10.A] F1 donors. The neonatally treated mice accepted skin grafts from B10 donors. Spleen cells from tolerant animals did not respond by proliferation to tolerated B10 antigens in vitro. However, spleen cells from tolerant mice recognized specific (B10) antigens and synthesized mRNA for the inducible 55-kDa interleukin-2 receptor (IL-2R) as did cells from normal animals. Maintenance of this early phase of cell activation upon contact with tolerated antigens is direct evidence against clonal deletion as a mechanism, in this particular model of neonatally induced transplantation tolerance.