Morphofunctional status of the adrenal cortex in female silver foxes and the effect of domestication and photoperiodic conditions

Studies on 7-day-old and 8-month-old female silver foxes indicate that additional photostimulation affects to a various extent morphological and functional condition of the adrenal cortex in animals selected and unselected for the domestication type of behaviour. In relatively wild foxes, additional illumination stimulated the activity of the reticular zone, whereas in domesticated animals the same photoperiod increased the activity in the fascicular zone. It is suggested that selection of silver foxes for the domestication type of behaviour affects the sensitivity of the adrenals to external illumination.     

Effect of glucose on adrenocortical activity of newborn piglets.

Adrenocortical function was followed in newborn piglets fasted for 36 hours and in piglets given only glucose or glucoplastic amino acids during this period. The greatest increase in relative adrenal weight, blood plasma 17-hydroxycorticosteroid (17-OHCS) levels and in the production of 17-OHCS by pig adrenals in vitro as compared with suckled controls was found in fasted piglets followed by amino acid-treated animals. The latter showed no significant differences in blood glucose between the initial and final values. The administration of glucose produced a transient hyperglycemia, failed to prevent the inhibition of body growth, resulted in a reduction in liver weight similar to that seen in fasted animals, and had a marked suppressive effect on the elevation of adrenocortical function. There were no significant differences in the production of 17-OHCS by adrenals in vitro per kg body weight between the glucose-treated and suckled piglets.     

Effect of stage of anestrus on the induction of estrus by the dopamine agonist cabergoline in dogs

Beagle bitches were administered the dopamine D2 receptor agonist cabergoline in 3 groups of 5 animals each, starting on known days of the estrous cycle. Cabergoline treatment was started in either early anestrus (Days 93 to 108), mid-anestrus (Days 123 to 156), or late anestrus (Days 161 to 192) at doses of 5 ug/kg/d, per os, and was continued until the confirmation of induced proestrus or for 40 d. Reproductive parameters were compared with those in 5 control anestrous bitches (Days 90 to 150). In control bitches, the mean (+/- SEM) interval to the next proestrus (73+/-11 d) resulted in an interestrus interval (192+/-9 d) similar to that of the previous cycles (196+/-11 d). In 14 of the 15 cabergoline-treated bitches, the next proestrus occurred within 4 to 30 d, was premature in early and mid-anestrous bitches and developed with low variability within groups. The resulting intervals to proestrus in bitches treated with cabergoline in early anestrus (20+/-2 d), mid-anestrus (14+/-3 d) and late anestrus (6+/-1 d) resulted in interestrus intervals in those groups of 131+/-5, 166+/-7 and 196+/-2 d, respectively. In response to treatment, interestrus intervals were reduced (P<0.05) and more synchronous (P<0.05) in early and mid-anestrus bitches, and were more synchronous (P<0.05) in late-anestrous bitches compared with those of control bitches or those of the previous cycle. Periovulatory estradiol and progesterone profiles of induced cycles in treated bitches were similar to those of spontaneous cycles in control bitches. Four of 5 control bitches and 12 of the 14 responding cabergoline-treated bitches became pregnant and produced normal litters. Plasma prolactin concentrations at Days 2 and 5 of treatment (0.3+/-0.1 ng/mL) and at the onset of proestrus shortly before the end of treatment (0.4+/-0.1 ng/mL) were lower (P<0.05) than those present in anestrus prior to treatment (1.7+/-0.6 ng/mL) or in control bitches. Prolactin was also low at the onset of proestrus in control bitches (0.5+/-0.2 ng/mL). The results demonstrate that prolactin-lowering doses of the dopamine agonist cabergoline can terminate the normal obligate anestrus in dogs, and that the effect occurs more slowly in early anestrus than in mid or late anestrus.     

The postnatal ontogenetic characteristics of adrenal function in female water voles Arvicola terrestris with different fur colors

Studies have been made of the effect of colour mutation on the development of the adrenals in female aquatic voles. Colour mutation in homozygote results in retarded growth rate of the whole body and the adrenals, as well as in postnatal changes in the dynamics of corticosteron content in the blood. Obviously, the effect of colour mutation on plasma concentrations of corticosteron is realised mainly via extra-adrenal mechanisms since no differences were found in corticosteron production by the adrenals and in the adrenal reaction to ACTH (5 U/g of the adrenals) in vitro in growing aquatic voles. During postnatal life, mutant black females exhibited lower sensitivity of the adrenals to the inhibitory influence of estradiol (40 ng/100 mg of the adrenals) as compared to wild phenotype animals.     

Cortisol production in fetal adrenals of the silver fox

The present study was designed to examine cortisol production by the silver fox fetal adrenals and their response to ACTH at different periods of prenatal life. Serum levels of cortisol were determined on days 35, 40, 45 and 50 of gestation (term on day 52) in embryos of both sexes. Cortisol content in adrenal tissue homogenates and its in vitro adrenal production were also investigated at the same time points. Hormones were measured by RIA. The levels of cortisol changed slightly during embryonic life. The adrenal content and the in vitro production of cortisol increased sharply and progressively (by 4-10 times from days 35 to 50 of gestation). The rises in cortisol content and in vitro production were associated with a rapid growth of the fetal adrenals. There were no sex differences in cortisol level and its adrenal content. ACTH (50 mIU per sample) increased the in vitro cortisol production by the adrenals in the two sexes on all the studied days (by 3-4 times on day 35 and by 1.4-1.7 times on day 50). These results indicate that 1) silver fox fetal adrenals are capable of synthesising cortisol; 2) ACTH is a potent activator of the in vitro cortisol production during embryonic life in this species.     

Increased LH pulse frequency and estrogen secretion associated with termination of anestrus followed by enhancement of uterine estrogen receptor gene expression in the beagle bitch

The relationships among pulsatile LH secretion pattern, estrogen secretion, and expression of the uterine estrogen receptor gene were examined throughout the estrous cycle in beagle bitches. In Experiment 1, blood samples were collected from 30 bitches every 10 min for 8 h from a cephalic vein during different phases of the estrous cycle. An increase in the mean plasma levels of LH occurred from mid to late anestrus (P < 0.01). The LH pulse frequency increased (P < 0.01) from late anestrus to proestrus, and was strongly correlated (r = 0.96, P < 0.001) with the mean plasma level of estradiol-17 beta (E2). In Experiment 2, middle uterine samples, including the myometrium and endometrium, from 18 bitches were taken at 6 stages of the estrous cycle. The total number of estrogen receptors and nuclear estrogen receptor and its mRNA levels in the uterus also increased (P < 0.01) from late anestrus to proestrus. Mean plasma E2 level and the number of uterine estrogen receptor were positively correlated (r = 0.81, P < 0.05). In Experiment 3, nine bitches were ovariectomized in mid anestrus. Two weeks later they received a single injection of 10 or 50 micrograms/kg, i.m., estradiol benzoate. The number of uterine estrogen receptor and their mRNA levels for ovariectomized bitches were low, but increased (P < 0.05) after treatment with a low dose of estradiol benzoate. These results suggest that increases in LH pulse frequency and estrogen secretion are associated with termination of anestrus and that subsequent enhancement of uterine estrogen receptor expression may be up-regulated by estradiol.     

Actions of acetyl-L-carnitine on the hypothalamo-pituitary-gonadal system in female rats.

Acetyl-L-carnitine (ALC) is known to affect several aspects of neuronal activity. To evaluate the neuroendocrine actions of this compound, several endocrinological parameters were followed in ALC-treated and control animals during recovery from dark-induced anestrus. In treated animals, serum luteinizing hormone (LH) and prolactin levels were higher than those of controls during the proestrous and estrous phases of the cycle, and serum estradiol levels were higher during estrus. No significant changes were observed in serum levels of follicle-stimulating hormone and progesterone. Uterine weight was increased in ALC-treated rats during proestrus and estrus, but not in diestrus. The basal release of gonadotropin-releasing hormone (GnRH) from perifused hypothalamic slices of ALC-treated animals was elevated at proestrus and diestrus, and GnRH release elicited by high K+ was higher during all three phases of the cycle. The basal release of LH from perifused pituitaries of treated animals was elevated in diestrus, and the LH response to GnRH was higher in estrus and diestrus I. Depolarization with K+ caused increased LH secretion during proestrus and estrus in treated animals. In contrast to these effects of ALC treatment in vivo, no direct effects of ALC were observed during short- or long-term treatment of cultured pituitary cells. These results indicate that ALC treatment influences hypothalamo-pituitary function in a cycle stage-dependent manner, and increases the secretory activity of gonadotrophs and lactotrophs. Since no effects of ALC on basal and agonist-induced secretory responses of gonadotrophs were observed in vitro, it is probable that its effects on gonadotropin release are related to enhancement of GnRH neuronal function in the hypothalamus.     

Functional characteristics of the adrenals in the ontogeny of male water voles Arvicola terrestris with various fur colorings

Studies have been made of the effect of colour mutations on the adrenal function in growing male voles. In postnatal life, the level of 11-oxycorticosteroids in the blood exhibited similar changes in brown (wild phenotype) and black (mutant) voles. The highest level was found in 1-2-month animals; a decrease took place to the 6th month which was followed by a secondary increase at the age of 8 months. Corticosteron production in vitro is the highest in brown males 5-month. In black voles, after 2 months the production remains unchanged, being rather high. In 5-, 6- and 8-month voles, corticosteron production in vitro increased, but not decreased after prior incubation. In mutant voles, the adrenals are more sensitive to stimulating effect of ACTH (5 units per 1 g of the adrenals) and inhibitory effect of estradiol (40 ng/100 mg) in vitro.     

Influence of morphine treatment in pregnant rats on the mineralocorticoid activity of the adrenals in their neonates

Exposure of pregnant rats to morphine, from day 11 to day 18 of gestation, was previously reported to induce both an adrenal atrophy and hypoactivity of the glucocorticoid function in newborns at term, but did not affect, in vitro, the responsiveness of those glands to adrenocorticotrophin hormone (ACTH) concerning corticosterone release. Moreover, these effects were mediated by maternal hormones from the adrenal glands. In the present work, we investigated the effects of a prenatal morphine exposure on the mineralocorticoid activity of the adrenals in neonates. The first aim of the present study was to determine in these newborns 1) the adrenal and plasma aldosterone concentrations at birth time and during the early postnatal period 2) the plasma levels of Na+ and K+ at birth time, 3) the in vitro responsiveness of the newborn adrenals to angiotensin II (A(II)) and ACTH. The second aim of our study was to investigate the mineralocorticoid activity of the adrenals in newborns from adrenalectomized mothers treated with morphine during gestation. According to present data morphine given to intact mothers induced in newborns a severe adrenal atrophy but increased adrenal aldosterone content and plasma aldosterone level. However, prenatal morphine was unable to affect significantly Na+/K+ ratio in both mothers and newborns. In vitro, the adrenals of neonates from morphine-treated mothers were unresponsive to An and ACTH for promoting aldosterone release; in contrast, aldosterone secretion was significantly stimulated by high potassium levels (55 mEq). Maternal adrenalectomy performed one day before the beginning of morphine treatment prevented morphine-induced adrenal atrophy but was unable to affect significantly the adrenal mineralocorticoid function of the offspring. Such data suggest that a prenatal morphine exposure stimulated both aldosterone synthesis and release in neonates. However, this basal hyperfunction did not appear to be coupled with an enhanced adrenal responsivity to AII or ACTH. Prenatal morphine-induced hyperactivity of the mineralocorticoid function of the newborn adrenals, which drastically contrast with hypoactivity of the glucocorticoid one, was independent of adrenal factors from maternal origin.     

Influence of reproductive status on in vitro oocyte maturation in dogs

In the bitch, oocytes need 48-72 h to complete post-ovulatory maturation to the metaphase II stage in the isthmus of the oviduct, an interval similar to that found in in vitro studies. The effect of estrous cycle stage on in vitro meiotic competence of dog oocytes has been described in several studies. However, there are no reports evaluating the possible effects of pyometra or pregnancy on subsequent potential of oocytes recovered from such females to undergo in vitro maturation.In this study, immature cumulus-oocyte complexes (COCs) were recovered from fresh excised domestic dog ovaries in various reproductive states. The donor females were classified into groups based on stage of the estrous cycle: follicular (proestrus or estrus), luteal (diestrus) or anestrus or at the clinical conditions of pregnancy and pyometra. Grades 1 and 2 oocytes were cultured in vitro at 37 degrees C in TCM-199, supplemented with 25 mM Hepes/l (v/v), and with 10% heat inactived estrous cow serum (ECS), 50 microg/ml gentamicin, 2.2 mg/ml sodium carbonate, 22 microg/ml pyruvic acid, 1.0 microg/ml estradiol, 0.5 microg/ml FSH and 0.03 IU/ml hCG. The nuclear maturation rate was evaluated at 72 h of incubation under Hoechst 33342 (10 microg/ml) staining for fluorescence microscopy. There was no statistical difference in nuclear progression to the MII stage among the various reproductive states (follicular phase, 5.4%; diestrus, 4.2%; anestrus, 4.4%; pyometra, 8.1% and pregnancy, 4.7%). Resumption of meiosis was 24.6% at the follicular phase, 19.6% for diestrus, 16.4% for anestrus, 37.1% for pyometra and 29.2% for pregnancy. Positive and higher numbers of residue above the expected value were observed for the pyometra and pregnancy conditions at the metaphase/anaphase I (MI/AI) stages.Our results indicate that in vitro nuclear maturation of dogs oocytes is not influenced by the in vivo reproductive status of the female. The quality of the oocyte is a more reliable indicator of its potential for meiotic maturation in vitro than the hormonal environment of the donor female at the time of oocyte retrieval.     

The estrous cycle in the dog: a review

Clinical, morphologic and hormonal changes throughout the estrous cycle in the dog are discussed in relation to the well established stages in this species — proestrus, estrus, metestrus and anestrus. Although most efforts in the past have been devoted to the period of reproduction, events throughout the remaining months of the cycle are presented to characterize the dog as a monocyclic animal in contrast to polycyclic species.     

Progesterone secretion by adrenal glands of hamsters and comparison of ACTH influence in rats and hamsters.

Studies were designed to determine a) if adrenal glands of hamsters secrete progesterone (PROG), b) the effects of adrenocritocotropin (ACTH) administration on adrenocortial function of rats and hamsters under the surgical conditions necessary for collection of adrenal venous blood from the left renal vein, and c) the effects of blood loss during sample collection. PROG was quantitated by the competitive protein-binding method after extraction and separation by sephadex LH-20 column chromatography. The presence of interfering quantities of androstenedione necessitated two column chromatographic steps. Glucocorticoids (11-OHCS) were determined fluorometrically. PROG was detected in adrenal venous plasma of female hamsters. The PROG concentration and secretory rate were 91 +/- 12 ng/ml and 4 +/- 1 ng/min, respectively, while the peripheral plasma level of the same animals was 2 +/- 0.2 ng/ml, indicating that the adrenal glands of female hamsters are capable of secreting PROG. ACTH administration increased PROG secretory rates in both hamsters (3 +/- 1 to 14 +/- 3 ng/min) and rats (62 +/- 9 to 152 +/- 32 ng/min) on estrus, as well as increasing the 11-OHCS secretory rate of hamsters (16 +/- 1 to 33 +/- 4 ng/min), but not of rats. The greater increase in PRCC than in 11-OHCS secretion may be related to excess PROG formation relative to the capacity of the 17alpha- or 21-hydroxylating enzyme systems. The adrenal venous PROG concentration and secretory rate of female hamsters infused with 10% dextran while collecting adrenal venous blood did not differ significantly from those of the non-infused animals, suggesting that this amount of blood loss (1 ml) does not influence PROG secretion.     

Production and secretion of C-19 steroids by rat and guinea pig adrenals

The concentrations of C-19 steroids were measured in guinea pig and rat adrenals before and after castration as well as after stimulation with adrenocorticotropin hormone (ACTH). Characterization of adrenal C-19 steroids was also carried out by isolation with high-performance liquid chromatography and gas chromatography/mass spectrometry (GC/MS). From radioimmunoassay (RIA) data, androstenedione (4-DIONE) and 11 beta hydroxyandrostenedione (11 beta-DIONE) were the major C-19 steroids found in guinea pig adrenals, and castration induced a decrease of 4-DIONE levels only while all other C-19 steroids remained unchanged. In rat adrenals, the major C-19 steroids were 4-DIONE and testosterone, and they were also markedly inhibited after castration. With the exception of 11 beta-DIONE, all other C-19 steroids in circulation were eliminated after castration in both animals species. After ACTH administration in the guinea pig, adrenal 4-DIONE and 11 beta-DIONE levels were markedly stimulated, while an increase of only 11 beta-DIONE was observed in plasma. In the rat, ACTH had a small stimulatory effect on adrenal 52-androstane-3 alpha, 17 beta-diol (3 alpha-DIOL) and plasma 11 beta-DIONE levels. Analysis of guinea pig adrenal steroids by GC/MS confirmed the presence of C-19 steroids in adrenals (namely, 4-DIONE and 11 beta-DIONE) while, in the rat, this could not be confirmed. Our data indicate that production of C-19 steroids occurs in guinea pig adrenals, and 11 beta-DIONE is the major C-19 steroid as well as the only C-19 steroid secreted into the circulation. In the rat, the production of C-19 steroids detected by RIA is not supported by GC/MS data.     

Stress- and (1-24)ACTH-induced alterations in adrenal and testicular steroidogenesis in Mongolian gerbils (Meriones unguiculatus): comparison of plasma levels, tissue content and in vitro secretion

Plasma glucocorticosteroid levels were significantly elevated 1 hr after confinement stress or (1-24)ACTH administration. Both adrenal content and in vitro secretion of glucocorticosteroids and progesterone from adrenals of stressed or (1-24)ACTH-injected animals were higher than values measured in controls. Neither adrenal testosterone content nor output of testosterone or progesterone from superfused testes were changed. Significant correlations were obtained between glucocorticosteroid plasma levels and corresponding adrenal content/in vitro secretion, adrenal progesterone content and output, and between adrenal glucocorticosteroid and progesterone content.     

Cortisol in fetal fluids and the fetal adrenal at parturition in the tammar wallaby (Macropus eugenii)

Glucocorticoid hormones may play a critical role in initiating parturition in tammar wallabies. In this study, we investigated the concentration of cortisol in fetal fluids and cortisol production by fetal adrenals over the last 3 days of the 26-day pregnancy and within 24 h postpartum. The fetal adrenals almost doubled in size between Days 24 and 26 of pregnancy, and their cortisol content increased over 10-fold during this period, from 10 pg to over 100 pg per adrenal pair. After birth, neonatal adrenals continued to grow, but cortisol content fell dramatically to 20 pg. The prepartum increase in adrenal cortisol was reflected by a substantial rise in cortisol concentrations in yolk sac fluid, allantoic fluid, and fetal blood, which were below 10 ng/ml on Day 24 and rose to over 40 ng/ml by Day 26. Cortisol concentrations in neonatal blood decreased postpartum, mirroring decreased cortisol content in neonatal adrenals. Cortisol production by the fetal adrenal was stimulated in vitro by ACTH and prostaglandin E2, suggesting that the in vivo increase may be stimulated by release of ACTH from the fetal hypothalamic-pituitary axis and prostaglandin E2 from the placenta. These results indicate that increasing cortisol production by the fetal adrenal is a characteristic of late pregnancy in the tammar wallaby and support the suggestion that fetal cortisol may trigger the initiation of parturition in this marsupial species.     

Effects of reproductive state on concentrations of thyroxine, 3,5,3'-triiodothyronine and cortisol in serum of dogs

Reproductive state of animals frequently is overlooked when examining endocrine functions of the thyroid gland and adrenal cortex. This experiment was done to determine effects of reproductive state on basal and stimulated concentrations of thyroxine (total T4), 3,5,3'-triiodothyronine (total T3) and cortisol in serum of adult Beagle dogs. Five male, 5 anestrous, 5 proestrous, 5 diestrous, 5 pregnant and 5 lactating dogs were fasted for 18 h before each dog received 5 IU of thyrotropic hormone (TSH) i.v. and 2.2 IU/kg of body weight of adrenocorticotropic hormone (ACTH) i.m. Blood samples were collected via jugular cannulas or by jugular venipuncture at 60, 45, 30, 15 and 1 min before and 0.5, 1, 2, 3, 4, 5, 8, 12 and 24 h after injection. Concentrations of T4 were similar in serum from diestrous and pregnant bitches but were greater (P less than 0.025) than those in dogs of other reproductive states before and after treatment with TSH. Concentrations of T3 were greater (P less than 0.005) in serum from diestrous bitches before and after TSH injection than in serum from dogs of all other reproductive states. Concentrations of T3 in males, anestrus, proestrus, pregnancy and lactation did not differ. Basal concentrations of cortisol did not differ consistently among reproductive states. However, concentrations post-ACTH were different (P less than 0.05) with anestrus = diestrus greater than lactation = pregnancy = male greater than proestrus. These results indicate that reproductive state of experimental animals must be considered when studying thyroidal and adrenal functions.     

Opiate antagonist counteracts reproductive inhibition by porcine ACTH extract.

The effects of naloxone, an opiate receptor antagonist, on the maturation of female mice chronically pretreated with ACTH from weaning, were examined. Naloxone treatment, begun at 40 days of age, blocked ACTH-induced delay of puberty. Naloxone also reversed decreases in weights of the reproductive organs produced by ACTH. Naloxone treatment also resulted in estrous cycling and increased the number of ACTH-treated mice with ovarian corpora lutea. These changes are reflected in increased LH levels in ACTH-naloxone treated vs. ACTH-saline treated animals. Naloxone alone may increase reproductive function. Effects of ACTH on spleen and thymus weights, presumably via the adrenals, were partially reversed by naloxone. ACTH may have a different mechanism in adrenal function as compared to reproductive function.     

Effect of dexamethasone on the estrous cycle length in Black Bengal goats (Capra hircus )

In an experiment to examine the relationships between adrenals and reproductive cycle, 10 mg dexamethasone (a synthetic glucocorticoid) were injected intramuscularly twice daily for 10 d to four Black Bengal goats, beginning on Day 11 of the synchronized estrous cycle. The extended length of the sexual cycle was monitored by the clinical signs of anestrus. Laparotomy was performed to examine the status of the ovary of an 8, 9, 10 and 8 d extended cycle, respectively. The length of the next cycle was normal. Endogenous cortisol values were suppressed for 11, 13, 20 and 24 d, respectively. It is thought that dexamethasone caused prolonged luteal function either by the suppression of prostaglandin F(2)alpha synthesis or by the suppression of pituitary stimulation of follicular growth.     

Steroid production by quartered and capsular rat adrenal gland in response to haemorrhage.

60 min after rapid bleeding (1.5--2.0 per cent of b. w.) both aldosterone and corticosterone production rate by quartered rat adrenals were found to be elevated. However, no difference was observed in the rate of aldosterone and corticosterone production by capsular adrenals of sham operated and hypovolaemic rats. Corticosterone production rate by decapsulated adrenals was much more higher after haemorrhage than in the control group. The same alterations could be observed incubating adrenal tissue with ACTH (0.3 mug per ml). Steroid production rate by quartered adrenals of sodium deficient rats was not affected by high in vitro concentration of angiotensin II (2.5 mug per ml). It is concluded that the effect of acute blood loss on corticosteroid biosynthesis of the rat is mediated by ACTH alone.     

Stage of the estrous cycle at the time of pregnant mare's serum gonadotropin injection affects pre-implantation embryo development in vitro in the mouse

The present study aims to analyze in the mouse the effect of the stage of the estrous cycle at the time of pregnant mare's serum gonadotropin (PMSG) injection on fertilization of ovulated cumulus-enclosed oocytes and later embryo development in vitro to the blastocyst stage. Quality of blastocysts was evaluated by staining and counting of total number of nuclei, mitotic index, percentage of apoptotic nuclei, and cell allocation to the inner cell mass (ICM) and trophectoderm (TE) lineage. Superovulation of hybrid (C57Bl/6JIco female x CBA/JIco male) female mice of 4-6 weeks of age was induced by a priming injection of PMSG at different stages of the estrous cycle followed after a 48-hr interval by human chrorionic gonadotropin. Our data indicate that injection of PMSG at the estrus phase gives the best outcome whereas injection of PMSG at the diestrus-1 or diestrus-2 phase provides the worst results. In fact, (1) total number of oocytes ovulated, number of ovulated oocytes enclosed by cumulus cells, and number of TE cells in day-5 blastocysts were significantly lower in diestrus-1 females than in estrus, diestrus-2 and proestrus mice; (2) percentage of day-5 blastocysts and total number of cells in day-5 blastocysts were lower in diestrus-1 and diestrus-2 females than in estrus and proestrus mice; and (3) percentage of apoptotic nuclei in day-5 blastocysts was lower in estrus mice than in diestrus-1, diestrus-2, or proestrus females. These data endorse previous studies suggesting that administration of gonadotropins in mice should be synchronized with the innate estrous cycle of females.