SUMMARY

1. The injection of 1 i.u. pregnant mares' serum (PMS) followed after 40 hr by 2 i.u. human chorionic gonadotrophin (HCG), or of 3 i.u. PMS followed by 3 i.u. HCG into mature female mice selected at random with regard to their oestrous cycle induces oestrus and mating in approximately 75%, and ovulation in 99% of them.

2. The induction of superovulation depends on the amount of PMS injected and on the strain of mice used.

3. Two types of egg are ovulated, one being normal and with a cumulus, the other degenerated and without cumulus. 93% of the normal eggs were fertilized and 98% of the pronucleate eggs possessed two pronuclei.

4. Approximately three-quarters of the females which mate in response to the injected gonadotrophins become pregnant, although this number was less than the number becoming pregnant after mating during natural oestrus. Many of the treated females carried their embryos to term and some gave birth to large litters, although resorptions, irregular distribution of embryos in the uterus, and difficulty during parturition occurred in some females. Mean litter size of the treated females was similar to that found after natural mating.

5. After more than one treatment with gonadotrophins, fewer females mated, ovulated, and became pregnant than after the first treatment. This reduction in response may have been due to the greater age of the females or to their decreased sensitivity to the hormones.

6. The value of the method as a technique for inducing oestrus, ovulation, and pregnancy in mature female mice is considered.

SUMMARY

The effects of exogenous oestrogen and progesterone on implantation and foetal mortality were studied in mice following induced superovulation with gonadotrophins.

Short-term treatment with progesterone during early pregnancy did not influence implantation or foetal mortality, but 2 mg daily from days 2 to 12 reduced the number of embryos that implanted and increased foetal mortality after implantation. These effects of progesterone were more severe when few embryos were implanted.

Oestradiol benzoate (OB), given after mating, reduced fertility by decreasing the proportion of mice with implanted embryos, causing cornification of the vagina in many mice during pregnancy. Administration of progesterone with OB did not alleviate these effects.

Administration of OB before superovulation, or lengthening the interval between the injections of PMS and HCG in order to extend the period of uterine stimulation by endogenous oestrogen, interfered with ovulation in some mice. Approx. one-quarter of the mice given oestrogen had cornified vaginal smears during early pregnancy. Both treatments decreased the numbers of mice with implanted embryos.

SUMMARY

The effects of an injection of pregnant mares' serum (PMS), human chorionic gonadotrophin (HCG), or of superovulation treatment with both of these gonadotrophins on subsequent reproductive behaviour of adult mice and on their response to a second superovulation treatment given shortly afterwards have been studied.

An injection of PMS had induced ovulation, but not superovulation, in dioestrous mice autopsied 24 hr later. The injection also induced oestrus and superovulation in many mice (c. 30%) 60–62 hr later, a positive correlation being found between mating and ovulation in these mice. The resumption of the natural oestrous cycle after an injection of PMS was delayed in some of the mice. HCG had induced ovulation in mice in metoestrus-II and dioestrus autopsied 24 hr later. Most mice quickly resumed their natural oestrous cycle after the injection of HCG.

If mice were not paired with males after the full superovulation treatment with PMS and HCG, they resumed their natural oestrous cycle within 3–6 days. Ovulation, fertilization and pregnancy during or after this natural oestrus were quite normal. These mice were also capable of further induced oestrus and superovulation when given a second course 1–3 days after the first treatment, though the number of mice that ovulated was lower, and the variability between mice in the number of eggs ovulated was higher, if the second treatment was given 1–2 days after the first.

The corpora lutea formed in mice that mated after a superovulation treatment were fully active. A second treatment induced superovulation in most mice, but failed to induce oestrus in the majority of them, especially when given during mid-pseudopregnancy. Fertilization and embryonic development were quite normal in the few mice that mated.

SUMMARY

The stages of the maturation divisions, ovulation, fertilization and the first cleavage in the eggs of two strains of adult mice have been timed following superovulation treatment. Superovulation was induced by a priming injection of pregnant mares' serum followed after a 40 hr interval by human chorionic gonadotrophin (HCG).

The oocytes were in the dictyate (germinal vesicle) stage until 2 hr after the injection of HCG. They then completed the prophase of the first maturation division, and the first metaphase plates were found 30 min later. From approx. 4½ to 8 hr all oocytes were in metaphase. The first maturation division and extrusion of the first polar body were then rapidly completed just before ovulation.

Ovulation began, as judged by the presence of eggs with adherent cumulus in the uterine tubes, 11 hr after the injection of HCG and was virtually complete by 14 hr. One strain of mice ovulated slightly later than another strain. The time at which ovulation occurred was very similar in mated and unmated females.

Fertilization and the first cleavage were studied in females which mated before ovulation began, and the mean intervals between various stages were as follows. Spermatozoa penetrated through the cumulus and zona pellucida in approx. 1 hr, and remained in the perivitelline space for 50 min. Pronuclei were formed 4¼ hr later, and the first cleavage division occurred at approx. 25 hr after penetration of spermatozoa into the eggs. These estimates are similar to those reported by other workers.

The formation of oocytes in the adult mammalian ovary, the timing of early stages in the prophase of the first maturation division, and the relation between natural and induced ovulation are discussed.

SUMMARY

Superfoetation has been induced experimentally in the mouse by treating females with gonadotrophins twice within a short period. Successive ovulations resulted from the treatments. After each treatment with gonadotrophins the females were paired with males, or were artificially inseminated, the spermatozoa carrying distinctive gene markers so that the paternity of offspring could be identified. If females were mated after one treatment and artificially inseminated after the other, embryos differing in age by 2½-3 days could develop from conception to birth in the same female. No examples of superfoetation were found when females mated in response to both treatments or when the difference in age of embryos from the first and second treatments was 3½-4 days; few females, however, could be induced to mate in response to each treatment.

In the experiments in which the age difference of the embryos was 2½-3 days, the second treatment with gonadotrophins delayed the transit of the first set of embryos along the uterine tubes. This often resulted in the blastocysts from the first treatment and the recently ovulated pronucleate eggs from the second treatment being found together in the tubes. Both groups of embryos apparently then moved normally along the tubes, implanted in the uterus at or about the same time, and were probably born simultaneously. If the age difference was 3½-4 days, transit of the first set of embryos was not delayed and many of these embryos were destroyed in the uterus.

The incidence of successive ovulations and the response of the female tract to repeated injections of gonadotrophins, the duration of the fertilizing capacity of spermatozoa in the female tract, aspects of sperm transport, and the possibility of increasing the incidence of mixed litters are discussed.

ABSTRACT

Ethane-1,2-dimethanesulphonate (EDS) destroys Leydig cells in the testis of the adult rat and subsequently a new population of Leydig cells develops. It has been reported that EDS is not cytocidal to the new immature Leydig cell population. In the present study, the effect of increasing the time-interval between injections of EDS on cytotoxicity to Leydig cells was examined. At time-intervals of 4–10 weeks between injections the response was similar to that seen after a single injection of EDS to the adult rat. Four days after the second injection, EDS was found to reduce substantially serum testosterone concentrations and in-vitro binding of ¹²⁵I-labelled human chorionic gonadotrophin (hCG) to testicular LH receptors which can be correlated with Leydig cell destruction. However, when the interval was only 2 or 3 weeks there was no reduction in serum testosterone, and ¹²⁵I-labelled hCG binding was not so markedly reduced. During days 1–6 after a second injection of EDS, administered 3 weeks after the first, there were marked reductions in serum testosterone concentrations and in ¹²⁵I-labelled hCG binding to testis homogenates within 24 h. Recovery from the effects of EDS was rapid, and increased Leydig cell activity was seen from 2 to 6 days after injection. In contrast to the established changes in the adult rat, there was only a 50% reduction in the number of Leydig cells positive for 3β-hydroxysteroid dehydrogenase 2 days after the second injection of EDS, and after 6 days the number of cells had increased. These experiments show that the immature Leydig cell of the rat is sensitive to the cytotoxic effects of EDS but that the temporal changes in Leydig cell activity after EDS treatment are different in developing and mature Leydig cell populations. The data are consistent with the view that EDS is preferentially cytotoxic towards steroidogenically active Leydig cells, allowing the resident population of precursor cells to continue to respond to the prevailing homeostatic mechanisms.

Journal of Endocrinology (1989) 123, 197–203

SUMMARY

A solid phase radioimmunoassay for glucagon was specially modified in order to overcome the problems involved in the measurement of glucagon release from incubated pieces of pancreas. The modified immunoassay procedure was used to study glucagon release from pieces of pancreas taken from newborn rats aged from 1 to 20 days. The glucagon content of rat pancreas was also measured during this period.

It was found that glucagon release from rat pancreas was stimulated by arginine and inhibited by octanoic acid at 1 and 2 days of age. However, glucagon release at 3 days of age was low, and between 3 and 7 days of age glucagon release could not be inhibited by octanoic acid or stimulated by arginine. At 10 and 20 days of age, the stimulatory action of arginine and the inhibitory action of octanoic acid were again noted. Glucagon release, measured at several ages, was not significantly affected by changes in glucose concentration. The glucagon content of the rat pancreas rose to a maximum at 5 days of age and then decreased gradually over a period of 90 days.

It is suggested that the low rate of glucagon release between 3 and 7 days of age may be a result of the high levels of blood fatty acids and ketone bodies found in the rat during this period.

ABSTRACT

A single injection of ethane-1,2-dimethanesulphonate (EDS; 100 mg/kg) selectively destroys Leydig cells in the testis of the adult rat; however, unconfirmed reports indicate that Leydig cells in the immature rat are not affected. In this study the effect of EDS was examined 2 days after treatment of rats aged 20, 25 or 35 days. There was a large reduction in the in-vitro binding of ¹²⁵I-labelled human chorionic gonadotrophin (hCG) to the homogenates of testes from EDS-treated immature rats. EDS reduced the testosterone content of the testes at all ages studied, but 2 days after injection had only significantly lowered the serum testosterone concentration of 25- or 35-day-old animals. Light microscopic examination of the testis of the 22-day-old rat, 2 days after treatment with EDS, indicated that there were still many cells staining for 3β-hydroxysteroid dehydrogenase. The interstitium also contained numerous atypical cells which did not stain for 3β-hydroxysteroid dehydrogenase. Electron microscopy of testes from the 22-day-old EDS-treated rat showed that Leydig cells were still present in the interstitium together with macrophages and fibroblast-like cells. Six days after EDS treatment of 20-day-old rats, but not 35-day-old rats, there was an increase in the binding of ¹²⁵I-labelled hCG to testis homogenate to 70% of control value. Testicular testosterone content 6 days after treatment of the 20-day-old rat had risen to 50% of the control testis value. These changes documented in the 20-day-old rat after EDS treatment can be explained by either a cytocidal effect with subsequent repopulation of new Leydig cells which has been described in the adult rat or by a reversible cytotoxic action which has not previously been documented.

J. Endocr. (1988) 119, 475–482

The rates of follicle growth and death have been estimated by a mathematical method in A, CBA, RIII and A × CBA strains of mice for which differential follicle counts were available. These rates were not uniform throughout life but were specific for the immature and mature phases of life. Significant heterogeneity of the rate estimates for particular follicle stages was also identified between strains and between intact and hypophysectomized mice, which explained the differing life-time patterns of follicular utilization in these animals.

ABSTRACT

The Leydig cells repopulating the adult rat testis after destruction by a single injection of the cytotoxic ethylene-1,2-dimethanesulphonate (EDS) were investigated. After 14 days, serum concentrations of LH and FSH were significantly raised and concentrations of testosterone in the serum and testis reduced. At 21 days, hormone concentrations had returned to within the normal range. Binding of ¹²⁵I-labelled human chorionic gonadotrophin (hCG) to testis homogenate, however, was still less than 10% of normal. After 21 or 28 days the ¹²⁵I-labelled hCG binding profiles of isolated Leydig cells from EDS-treated rats, separated on a Percoll gradient, showed a single peak similar to that of immature (25 days old) rats. After 49 days, ¹²⁵I-labelled hCG binding resolved into two peaks more like that of normal adult rats. Using a quantitative cytochemical method, 3β-hydroxysteroid dehydrogenase activity in individual Leydig cells of unfixed testis sections was determined. Activity was increased by 70% (P < 0·05) in repopulating Leydig cells 21 days after EDS treatment compared with cells from vehicle-treated rats. In addition, Leydig cells were still capable of further 'in-vivo' stimulation by pharmacological doses of hCG. These data indicate that Leydig cells repopulating the testis are homogenous. Fewer cells from the newly formed population are capable of maintaining normal serum concentrations of testosterone and must thus be individually more active in secreting testosterone. In these respects, the Leydig cells repopulating the adult rat testis after EDS treatment more closely resemble those of the fetal rat testis.

J. Endocr. (1988) 117, 11–18