Pattern of serum concentrations of prolactin and progesterone during pregnancy and lactation in the brown hare (Lepus europaeus).

A heterologous radioimmunoassay system developed for the rabbit has been shown to measure prolactin in the hare. Concentrations of prolactin showed a significant (P less than 0.01) increase in the last 3 days of pregnancy (87.7 +/- 11.7 compared with 9.8 +/- 1.4 (S.E.M.) micrograms/l; n = 10 and 9 respectively) in pregnant females isolated from males, as well as in pregnant females kept with males and mating prepartum. This rise of prolactin at the end of pregnancy was not due to mating stimuli and occurred at a time when progesterone levels were still high (159 nmol/l). The injection of a slow-release preparation of bromocriptine (5 mg s.c.), which reduces prolactin secretion at the end of pregnancy, did not impair parturition. During lactation, prolactin levels increased significantly (61.2 +/- 19.8 compared with 5.3 +/- 0.1 micrograms/l; P less than 0.01) only after suckling stimuli.

Annual variations of in vitro GNRH release by hypothalamic explants in intact and castrated male mink: relations with LH, FSH and testosterone circulating serum levels.

In mink, a short-day breeder, testis growth begins in autumn (November), reaches a maximum in February, before matings occur, and decreases from March to very low volumes during spring and summer. To study the effects of season and testosterone feedback on gonadotrophin and GnRH secretion, the annual variations of LH, FSH, testosterone and GnRH were studied in intact and castrated mink. As portal blood sampling raised serious difficulties, an in vitro static incubation system was used for studying GnRH variations. In intact mink, serum LH concentrations did not vary significantly throughout the year, whereas FSH concentrations increased significantly between September and November then decreased to a minimum in January. Testosterone values rose significantly from November to a maximum from January to March, decreased very rapidly thereafter. Castration in November resulted in a significant increase in LH and FSH concentrations which remained higher than the values measured in intact males throughout the year. In long-term castrated mink, FSH concentrations did not fluctuate during the year, whereas LH concentrations showed an annual variation, with high values in April and August. For the study of in vitro GnRH liberation, medio-basal hypothalamic explants were incubated in Krebs-Ringer phosphate buffer for 3 periods of 15 min, and stimulated with copper complexed equimolarly with histidine (Cu/His, 200 microM) and prostaglandin E2 (PGE2, 10 microM). After Cu/His, the release of GnRH was 1 to 4 fold the basal release; after PGE2, the increase was 4-7 fold the basal release. The basal release of GnRH increased significantly between September and October to reach a maximum in November, decreased significantly in December to a minimum in February then increased progressively from May. The release of GnRH stimulated by Cu/His and PGE2 showed the same seasonal variation as the basal release. Castration 8 days before the sacrifice did not alter the release of GnRH, except in December: the release stimulated by PGE2 was then higher in intact than in castrated mink. Taken together, these results indicate that, with an in vitro static incubation system, it is possible to study the annual variations of GnRH release and to correlate these variations with those of serum gonadotrophin and testosterone concentrations. The synthesis and release of GnRH increased slightly from May, under long days, then more rapidly from September, resulting in an increased secretion of FSH in October, responsible for testis recrudescence. The annual pattern of basal and stimulated GnRH release was similar in intact and castrated mink, suggesting a direct effect of the season on the hypothalamus, rather than a negative feedback effect of the testis; however, testosterone seemed to feedback mainly at the pituitary level.

Structural aspects of oocyte maturation in the blue fox (Alopex lagopus).

Blood samples were taken weekly from seventeen mature blue fox vixens (average age five years), from late anoestrus until pro-oestrus, and then taken daily. The vixens were sacrificed at various stages of oestrus, and oocytes were collected from ovarian follicles by aspiration, and/or from oviducts by flushing. The structural features of oocyte maturation were related to the time of the luteinizing hormone (LH) peak. On days 1-2 after the LH peak the oocyte nucleus migrated from a central to a peripheral position in the ooplasm and assumed a flattened appearance. The cumulus investment expanded simultaneously and ovulation took place around day 2. On days 2-3 the oocyte nuclear envelope broke down, the nucleoli disappeared, the metaphase of the first meiotic division was reached, the Golgi complexes decreased in size, the perivitelline space enlarged, and all junctional contact between cumulus cell projections and oocyte was disrupted. On days 3-5 the first polar body was extruded, the metaphase of the second meiotic division was reached, and the cumulus cells degenerated. On day 5 the release of cortical granule content was occasionally seen, and from day 6 the oocytes showed signs of degeneration. In a few animals deviant oocyte maturation was noticed.

Periovulatory endocrinology and oocyte maturation in unmated mature blue fox vixens (Alopex lagopus).

Nine of 10 mature blue fox vixens (Alopex lagopus) in spontaneous oestrus ovulated approximately 2 days after the preovulatory increase in luteinizing hormone (LH). Plasma concentrations of follicle-stimulating hormone and progesterone increased simultaneously with the LH peak, whereas oestradiol-17 beta peaked 1 day previously. In the tenth vixen, an LH peak was not observed, and neither visible follicles nor corpora lutea were found in the ovaries 6 days after peak vaginal electrical resistance. Eggs were ovulated as primary oocytes, but oocyte maturation was initiated within the day of ovulation (2 days after the LH peak). Within the next 2 days (3-4 days after the LH peak) the first polar body was extruded, and the cumulus mass was completely dissociated from the zona pellucida. The interval between the preovulatory LH peak and initiation of the final oocyte maturation is thus considerably longer in the blue fox than for example in the cow (48-72 h compared with 9-12 h). This suggests that the relationship between these two events is somewhat different in the blue fox.

Pituitary and ovarian responses to luteinizing-hormone-releasing hormone during pregnancy and after parturition in brown hares (Lepus europaeus).

Female hares were given an i.v. injection of 5 micrograms luteinizing-hormone-releasing hormone (LHRH) between Days 7 and 19 (n = 21), 20 and 33 (n = 17) and 34 and 41 (n = 17) of pregnancy, and in the 3 days after parturition (n = 16). Whatever the stage of pregnancy, the LHRH injection induced a release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and an acute secretion of progesterone; these hormonal responses increased significantly during pregnancy, to reach values similar to those observed in nonpregnant, nonpseudopregnant females during the breeding season in the 3 days after parturition. However, the release of LH remained monophasic in pregnant and post-partum females, in contrast to the unmated females during the reproductive season, in which there was a biphasic profile. The proportion of ovulating females after LHRH treatment was approximately 60% at the beginning and end of pregnancy; and, after parturition, fell to 23% between Days 20 and 33. After Day 33, the pituitary response to LHRH was significantly higher in ovulating than in nonovulating females. At the beginning of pregnancy, 67% of females aborted after LHRH injection; after Day 20, the incidence of abortion decreased significantly and was 0% from Day 34. The amplitude and duration of progesterone secretion by the new corpora lutea resulting from ovulation after LHRH injection were similar to those of corpora lutea induced in nonpregnant females during the breeding season.(ABSTRACT TRUNCATED AT 250 WORDS)

Gonadotrophins and sex steroids during pregnancy and natural superfoetation in captive brown hares (Lepus europaeus).

In brown hares, which are induced ovulators, sexual behaviour occurs episodically at the beginning of pregnancy. From Day 34 (length of pregnancy is 41 days), the frequency of sexual chases followed by mating, ovulation and fertilization increased and 59% of pregnant females presented a natural superfoetation. The pattern of circulating luteinizing hormone (LH), follicle-stimulating hormone (FSH), oestradiol and progesterone was studied in 13 pregnant females left permanently with a male, and in 10 females isolated from males around Day 20 of pregnancy. In the 2 groups FSH concentrations were high at the beginning and end of pregnancy. All females presented a peak value of FSH in the last 4 days of pregnancy, regardless of mating stimuli. This peak value was higher for females left permanently with a male than for isolated ones. Oestradiol concentrations fluctuated between 20 and 100 pg/ml, without any clear correlation with sexual behaviour, stage of pregnancy or profiles of other hormones. Prepartum matings occurred when progesterone values were still greater than 50 ng/ml; they were followed by a transient rise in LH and by a periovulatory progesterone secretion, with values above 100 ng/ml in the morning after mating. Such modifications of LH and progesterone were not detected before Day 34, suggesting that mating stimuli are not able to induce an LH surge at the beginning of pregnancy. After Day 34, mating can induce an LH surge, ovulation and superfoetation.

Oestrogen and progesterone concentrations in peripheral blood in pregnant red foxes (Vulpes vulpes).

Oestrogen levels were low during most of gestation, but there was a significant increase (P less than 0.05) in oestradiol concentrations at implantation. Early pregnancy was characterized by high levels of progesterone which decreased significantly (P less than 0.001) thereafter, but there was no decline in progesterone or rise in oestrogen levels at parturition. There was no difference in the length of progesterone secretion between pregnant and non-pregnant females.

Effects of melatonin treatment on the gonadotropin-releasing hormone neuronal system and on gonadotropin secretion in male mink, Mustela vison, in the presence or absence of testosterone feedback.

The effects of subcutaneous melatonin capsules on the gonadotropin-releasing hormone (GnRH) immunoreactive (ir) system and the secretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH) have been tested in intact, castrated, and castrated adult male mink supplemented with testosterone. Animals were transferred in July, i.e., during the period of sexual rest, under a daily light:dark cycle of 16-hr light and 8-hr darkness and studied over 13 weeks. GnRH (ir) perikarya, visualized by immunocytochemistry, were counted on serial coronal sections from the diagonal band of Broca to the infundibulum. Serum FSH and LH concentrations were measured by radioimmunoassay. In intact mink, melatonin induced a significant increase in the number of (ir) perikarya and in FSH and LH concentrations 3 and 8 weeks, respectively, after melatonin capsule implantation. In castrated mink, the number of perikarya and the concentrations of FSH, which had increased within 2 weeks after castration, did not change during melatonin treatment. In contrast, the concentration of LH, which were not altered by castration, increased 3-6 weeks after the onset of melatonin administration, suggesting a stimulation of GnRH release. In castrated testosterone-treated mink, the number of perikarya was increased as in castrated males, but the elevation of FSH in response to castration was prevented. Within 2 weeks after melatonin capsule implantation, the concentrations of FSH decreased while those of LH remained low, indicating an inhibition of GnRH release. These results show that testosterone modulates the effect of melatonin on the activity of the GnRH-gonadotropin system.

A case of granulosa cell tumour with cystic endometrial hyperplasia in a blue fox vixen (Alopex lagopus).

The clinical features of a case of granulosa cell tumour in the blue fox (Alopex lagopus) are described. The associated hormonal changes are discussed in relation to present knowledge of the regulation of the oestrous and furring cycles in this species.

Seasonal variations in the pituitary response to LHRH in the brown hare (Lepus europaeus).

In the brown hare, fertile mating takes place from the beginning of December to September. Pituitary and ovarian response to a monthly i.v. injection of 5 micrograms LHRH was studied from September 1983 to October 1984 in 2 groups of 6 hares. The basal concentrations of LH remained undetectable until the end of January, rose from 0.23 +/- 0.14 ng/ml from February to a maximum of 1.44 +/- 0.57 ng/ml in July. LHRH injection was always followed by a release of LH. Between September and December, the LH value peaked 15 min after injection and returned to basal concentrations 2 h later. From January, this pattern altered and a second peak of LH appeared 2 h after injection. Peak levels 15 min after LHRH were around 10 ng/ml between September and December, increased from 47.0 +/- 8.0 ng/ml in January to 106 +/- 33 ng/ml in July and decreased in August (69.4 +/- 10.6 ng/ml). The values of the second peak rose from 11.0 +/- 2.2 ng/ml in January to 90.6 +/- 12.4 ng/ml between March and July and decreased in August (24.5 +/- 5.1 ng/ml). The LH surge induced by LHRH was always followed by a transient rise in progesterone. During the breeding season, this progesterone secretion increased considerably. Ovulation was possible between January and August and the number of ovulating females was maximum between March and July. The amount and duration of progesterone secretion during the resulting pseudopregnancies increased during the breeding season.

Influence of season of birth on onset of gonadotrophic and ovarian functions in young doe hares (Lepus europaeus).

The pituitary and ovarian responses to a monthly i.v. injection of 5 micrograms luteinizing-hormone-releasing hormone (LHRH) were studied in three groups of young doe hares, born in January-February (group I), in April (group II) or at the end of the breeding season (August-September, group III). The LHRH injection was always followed by a release of LH and progesterone, which did not differ among the three groups at 3 months of age. The pituitary and ovarian responses to LHRH increased gradually from the age of 3 months in groups I and III and from the age of 9 months in group II. One female of the ten born in January-February ovulated and reached puberty in June, at the age of 4 months, but with a weak pituitary response. The females born in April displayed a seasonally delayed puberty, at 9 months of age (two of five females ovulated in the next January). Four of the five females born at the end of the breeding season ovulated after LHRH when 5 months old (in February), with a full pituitary-ovarian response. The low pituitary response of group I in June-August, even if 10-20% of females ovulated after LHRH, suggests a need for a period of short days. Then, the most favourable conditions for the hare to reach puberty would be a period of short decreasing daylengths during the fall, followed by increasing daylengths after the winter solstice.

Plasma androgen patterns during delayed implantation in the European badger (Meles meles L.).

Androstenedione and testosterone were measured by radioimmunoassay after chromatography on celite microcolumns throughout the delayed implantation period in the European badger (Meles meles L.). Androstenedione and testosterone concentrations varied from 0.05 to 22 ng/ml, and from 30 pg/ml to 359 pg/ml, respectively. There was a typical biphasic pattern in the fluctuation of these two steroids. Androstenedione levels increased between June and August (X= 7.9 +/- 0.8 ng/ml), decreased from September to November (X = 2.3 +/- 0.3 ng/ml), and then increased again from December to February (X = 6.1 +/- 0.7 ng/ml). The same fluctuations of the testosterone levels were observed throughout the diapause, but the concentrations were lower. The overall correlation between androstenedione and testosterone was highly significant (r = 0.75, P less than 0.001). There was also a similarity between the androgen and progesterone patterns of secretion (r = 0.31), particularly at the end of the delay period (r = 0.73, P less than 0.001). No correlation could be demonstrated between androgens and estrogens.

Patterns of plasma progesterone, androgen and oestrogen concentrations and in-vitro ovarian steroidogenesis during embryonic diapause and implantation in the mink (Mustela vison).

Peripheral plasma progesterone concentrations exhibited an increase 10 days before implantation, coinciding with the resumption of blastocyst growth and with a decrease in plasma androgen values (DHA, androstenedione, testosterone). No definite pattern of oestrone was observed and oestradiol concentrations remained undetectable. The production of steroids by dispersed luteal cells showed that the growth of the corpora lutea paralleled that of blastocysts and resulted in hypertrophy followed by hyperplasia of the luteal cell. The production of progesterone in the medium increased with blastocyst size up to implantation; it was enhanced by mink charcoal-treated serum, but prolactin, LH, FSH or a combination of these hormones did not affect the progesterone production, whatever the stage of diapause. DHA and androstenedione secretion increased in the two last stages of blastocyst growth and was enhanced by LH. The conversion of androstenedione and testosterone into oestrone and oestradiol was observed at all stages of embryonic diapause, indicating that corpora lutea contain aromatase activity even at an early stage. The secretion of oestrone was higher than that of oestradiol. The non-luteal tissue contributed up to 50% of the steroid production; while progesterone and androgen production remained constant, that of oestradiol decreased at the end of the delay period. These results indicated a change in the size and the secretory capacity of the luteal cell related to blastocyst development and implantation. Although progesterone was the main product of the corpora lutea, androgens and oestrogens were also secreted.

Endogenous circannual rhythms and photorefractoriness of testis activity, moult and prolactin concentrations in mink (Mustela vison).

Mink are seasonal photosensitive breeders; testis activity is triggered when days have less than 10 h light. Increasing and decreasing plasma concentrations of prolactin induce the spring and autumn moults. In a 5 year experiment, males were maintained under short days (8 h light:16 h dark) at 13 degrees C or long days (16 h light:8 h dark) at 21 degrees C, winter and summer conditions, respectively. Under winter and summer conditions, circannual cycles of prolactin secretion and moulting were observed at intervals of about 11 months. Recurrence of testis cycles was not evident. In a second experiment, males were maintained under an 8 h light:16 h dark cycle from the winter solstice or under 10 h light:14 h dark, 12 h light:12 h dark or 14 h light:10 h dark cycles from 10 February. Under 8 h light:16 h dark cycle, testis regression was slightly later than under natural conditions, indicating photorefractoriness. However, mink remained sensitive to light: the longer the photoperiod, the faster the testis regression. In a third experiment, males were transferred under 8 h light:16 h dark or 16 h light:8 h dark from 15 May (group 1), 12 June (group 2) or 4 July (group 3); males submitted to long days received melatonin capsules on the day of transfer. Increasing concentrations of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and testis volume were shown by half the males in group 2 and nearly all the males in group 3; the constant release of melatonin from implants was more efficient than short days; but in the three groups, prolactin concentrations decreased in the few days after short-day or melatonin treatment. Overall, the results demonstrate endogenous circannual rhythms of prolactin secretion, body weight and moulting. Although a refractory period to short days was observed, the annual cycle of testis activity totally relies on the annual changes in daylength.

Heterologous radioimmunoassay of fox LH: levels during the reproductive season and the anoestrus of the red fox (Vulpes vulpes L.).

A heterologous radioimmunoassay using ovine LH as the labeled hormone, canine LH as the standard, and an antiovine LH rabbit serum was validated for the measurement of fox LH. Physiological validation of the assay was evidenced by the high concentrations of LH at oestrus and following ovariectomy or the administration of LH-RH. Throughout the year, plasma LH levels demonstrate important variations, being low during and after the luteal phase (1.4 +/- 0.3 ng/ml) (mean +/- SE) and increasing during the second part of anoestrus (5.2 +/- 1.4 ng/ml). This latter increase might be correlated with that of androgens observed at the same period. Several LH rises preceded the preovulatory LH surge.

Fertilization and early embryonic development in the blue fox (Alopex lagopus).

A total of 15 blue fox vixens aged 1-6 years were mated, 12 once on the first day of estrus and three a second time 48 hr after the first mating, and were killed 4 hr to 8 days following mating. Ova were collected from the oviducts, evaluated by stereomicroscopy, and studied by transmission (TEM; N = 49, 12 vixens) or scanning (SEM, N = 11, three vixens) electron microscopy. At 0-3 days after ovulation, the ova had not cleaved and were at different stages of meiotic maturation. In about one-half of these ova, representing all stages of meiotic maturation, a decondensing sperm head without nuclear envelope or a small pronucleus with partial nuclear envelope was observed. No clear relationship was found between maternal meiotic stage and the stage of paternal pronucleus formation. Sperm tails were never identified in the ooplasm. Cortical granules were released after sperm penetration at early stages of meiotic maturation. Thus the block against polyspermic penetration was activated during maturation of the oocyte. The first two-cell stage appeared 4 days after ovulation (3 days after mating), the first four-cell stage the following day (day 5), and the first eight-cell stage 6 days after ovulation (5 days after mating). In a single vixen mated late (7 days postovulation) two- to four-cell stages appeared the following day (day 8). This indicates that the time required for the first cleavage division decreases with increasing interval from ovulation to mating.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid detection of the preovulatory luteinizing hormone peak in the blue fox (Alopex lagopus) by an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) developed for the rapid measurement of luteinizing hormone (LH) in blood of many mammalian species has been validated in the fox. For this purpose, daily plasma samples collected from six vixens during the periovulatory period were assayed for LH using ELISA. The results were compared with those obtained by radioimmunoassay (RIA), and related to parameters used to determine the time of ovulation (onset of oestrus, vaginal electrical resistance peak) and to ovarian findings. This ELISA provided a highly sensitive measure of LH (125 pg ml-1) from a 10 microliters plasma sample within 3 h. Plasma concentrations of LH measured by ELISA and RIA were significantly correlated (r = 0.911). In the blue fox, ovulation occurs once a year and coincides with the start of oestrus. The LH peak preceded the start of oestrus by 1.5 days (1-3) and preceded the vaginal electrical resistance peak by 2.2 days (1-4). The rapid and precise detection of the preovulatory LH peak by this assay provides a convenient method to determine the time of ovulation and therefore the optimum time for artificial insemination or oocyte collection for fertilization in vitro.

Fertilization in vitro of oocytes matured in vivo in the blue fox (Alopex lagopus).

Six unmated blue fox vixens were killed 2-3 days after peak vaginal electrical resistance. Measurements of luteinizing hormone (LH) showed that this corresponded to a time period of 6-10 days after the preovulatory LH peak in these vixens. A total of 63 ova were collected from the oviducts of the vixens, and 45 of the ova were used for study of fertilization in vitro. The ova were placed in 50 microliter droplets of fertilization medium and inseminated with 15,000-45,000 frozen-thawed silver fox spermatozoa that had been separated by swim-up technique, except for nine ova that served as controls for parthenogenesis. Only two of the 36 inseminated ova, both from a vixen killed 6 days after the preovulatory LH peak and inseminated with 15,000 spermatozoa, developed past the four-cell stage. One reached the morula stage 144 h after insemination. Despite a low success rate, the present experiment shows that fox ova matured in vivo may be fertilized and undergo initial embryonic development in vitro.

Relationship between gonadotrophins, inhibin and sex steroid secretion during the periovulatory period and the luteal phase in the blue fox (Alopex lagopus).

The blue fox is a monestrous seasonal breeder with a pro-oestrus and oestrus lasting several days and a prolonged luteal phase in non-pregnant as well as pregnant vixens. Ovulation occurs 2 days after the preovulatory luteinizing hormone (LH) peak at the onset of oestrus; oocytes are ovulated as primary oocytes and maturation is completed 2-3 days later. Fertilization is possible during all stages of oocyte maturation until degeneration of oocytes 5 or 6 days after the LH peak. In this study, changes in plasma concentrations of follicle-stimulating hormone (FSH) and immunoreactive inhibin (iINH) and their relationships with LH, oestradiol, androstenedione, testosterone and progesterone secretion and with the periovulatory events, were determined throughout the reproductive cycle of 14 vixens. In late anoestrus and pro-oestrus, FSH and iINH were inversely related to each other (r = -0.84, P < 0.001); whereas FSH concentrations declined to reach low values within the 5 days preceding the LH peak, iINH secretion increased, as did that of oestradiol and androgens. Thereafter, iINH and steroid concentrations with the exception of progesterone reached maximal values at the time of the LH and FSH peaks and decreased within the following 2-3 days in the early luteal phase. Progesterone started to increase simultaneously with the LH peak, and when oocyte maturation was completed (around day 4), progesterone concentrations were increasing, while those of other hormones were low. A postovulatory release of FSH was detected between days 6 and 10; it preceded the rise in iINH, which began on day 8 when concentrations of progesterone were already high and those of oestradiol and LH were low. Progesterone and iINH were positively correlated over the period day 6 to day 49 (r = 0.62, P < 0.01). These results indicate that, in the blue fox, iINH is secreted by the preovulatory follicle and the corpus luteum; that during pro-oestrus, iINH may act in synergy with oestradiol to reduce the secretion of FSH and that after the LH peak the decrease of iINH and oestradiol concentrations may be responsible for the postovulatory release of FSH, which in turn causes an increase in iINH, possibly secreted by the corpus luteum and, as a result, FSH secretion is suppressed.