Influence of prenatal stress on steroidogenesis in gonads of blue foxes

Handling is a source of stress for farm bred blue foxes. The influence of handling during the late gestational period was investigated in 10-day-old male and female blue foxes. Testosterone and estradiol were measured by RIA in the plasma, gonadal homogenates andin vitro incubates, from blue foxes of both sexes. The gonads were incubatedin vitro without or with human chorionic gonadotropin. In cubs of both sexes, the gonad weights and ovarian estradiol production were decreased by stress. The testicular testosterone and ovarian estradiol contents were increased in prenatally stressed cubs as compared to the controls. The testicular content and baselinein vitro production of testosterone were not affected by prenatal stress, but the testicular response to human chorionic gonadotropin was higher in the stressed group. This study suggests that prenatal stress induced by handling pregnant vixens may influence gonadal steroidogenesis and that this effect was more pronounced in female cubs.     

Effect of prenatal stress on the pituitary-adrenal axis in blue foxes

Handling is a source of stress for farm bred blue foxes. The influence of handling during the late gestation period on the pituitary--adrenal axis was studied in 10-day old male and female blue foxes. Cortisol and progesterone were measured by radioimmunoassay in the plasma, adrenal homogenates, and in vitro incubates from animals of both sexes. Adrenals were incubated in vitro in the absence or presence of ACTH. In addition, the adrenal weight and plasma concentration of ACTH were assessed. In cubs of both sexes, the adrenal weight was decreased after prenatal stress. The plasma concentration of progesterone and the adrenal cortisol in vitro production were elevated in the prenatally stressed female cubs, as compared to the control, along with the adrenal progesterone in vitro production in prenatally stressed male cubs. The adrenal cortisol and progesterone content and plasma ACTH and cortisol concentrations were not affected by prenatal stress. In conclusion, the results of this study suggest that the prenatal stress induced by handling pregnant vixens can affect the pituitary--adrenal axis in neonatal offspring, this effect being more pronounced in female cubs.     

Effect of Prenatal Stress on the Pituitary–Adrenal Axis in Blue Foxes

Handling is a source of stress for farm bred blue foxes. The influence of handling during the late gestation period on the pituitary–adrenal axis was studied in 10-day old male and female blue foxes. Cortisol and progesterone were measured by radioimmunoassay in the plasma, adrenal homogenates, and in vitro incubates from animals of both sexes. Adrenals were incubated in vitro in the absence or presence of ACTH. In addition, the adrenal weight and plasma concentration of ACTH were assessed. In cubs of both sexes, the adrenal weight was decreased after prenatal stress treatment. The plasma concentration of progesterone and the adrenal cortisol in vitro production were elevated in the prenatally stressed female cubs, as compared to the control, along with the adrenal progesterone in vitro production in prenatally stressed male cubs. The adrenal cortisol and progesterone content and plasma ACTH and cortisol concentrations were not affected by prenatal stress. In conclusion, the results of this study suggest that the prenatal stress induced by handling pregnant vixens can affect the pituitary–adrenal axis in neonatal offspring, this effect being more pronounced in female cubs.     

Mechanisms involved in the ability of human chorionic gonadotropin to increase the responsiveness of the infant rat's testis to follicle-stimulating hormone

Pretreatment of 9-day-old rats for 3 days with human chorionic gonadotropin (hCG) increases the amount of estradiol secreted by the testis in response to in vivo or in vitro stimulation with follicle-stimulating hormone (FSH). Potential mechanisms for this sensitizing effect were studied by treating infant rats with a variety of agents and then using radioimmunoassay to determine testicular estradiol secretion. Substitution of 3 days priming with estradiol for hCG did not enhance subsequent in vitro responsiveness to FSH. Subcutaneous capsules of 1,4,6-androstatriene-3,17-dione (ATD) blocked stimulation of testicular aromatization in vivo by hCG or FSH. ATD capsules alone, or when combined with the antiestrogen tamoxifen, were not able to alter the ability of hCG pretreatment to increase responsiveness to in vitro FSH. It was concluded that estradiol was not involved in the sensitization caused by hCG in this model system. When gonadal tissue from 12-day-old rats was incubated in the presence or absence of 0.6 microM testosterone and various concentrations of FSH, more estradiol was secreted by testes in the containing testosterone. The amount secreted was not different from that noted after hCG priming. Priming of 9-day-old rats for 3 days with the nonaromatizable androgen 5 alpha-dihydrotestosterone did not influence the amount of estradiol secreted in response to FSH. It is further concluded that hCG augments the testicular aromatization response of infant rats to FSH by providing additional substrate for these reactions.     

Impact of γ-hexachlorocyclohexane exposure on plasma gonadotropin levels and in vitro stimulation of gonadal steroid production by carp hypophyseal homogenate in Carassius auratus

Male and female Carassius auratus were exposed to safe (SC; 0.01 ppm) and sublethal (SL; 0·1 ppm) concentrations of an organochlorine pesticide γ-hexachlorocyclohexane (γ-HCH) for 4 weeks during the pre-spawning phase (June) of the annual reproductive cycle. Gonadosomatic index and gonadotropin levels were significantly lower after exposure to both γ-HCH concentrations than in control fish. After 4 weeks exposure, gonadal tissue from control, SC and SL exposed fish was incubated with carp hypophyseal homogenate (chh). The chh stimulated production of testosterone, 17,20β-dihydroxy-4-pregnen-3-one (17,20βP), 11-deoxycortisol, and 11-ketotesterone (11-KT) was estimated in the unconjugated (free) and conjugated (glucuronide) fractions by radioimmunoassay. In both sexes, testosterone production was greatly decreased in γ-HCH exposed fish compared to controls. 17,20βP production was low in all fish and was unaffected by γ-HCH. Free 11-deoxycortisol production by testicular fragments was higher in SL and SC compared with controls, while conjugated 11-deoxycortisol was increased only higher in the SL. In ovarian fragments from exposed fish, free 11-deoxycortisol decreased while glucuronide concentrations increased compared with controls. 11-KT production was significantly decreased in testicular fragments of exposed fish. The results indicate that γ-HCH inhibits gonadal recrudescence by decreasing both gonadotropin secretion and its potential for stimulation of steroidogenesis.     

In vitro production of ovarian steroids in yellow perch (Perca flavescens): effects of photothermal manipulation, gonadotropin and phorbol ester

We investigated the capacity of ovaries of yellow perch to produce steroid hormones in vitro and the ovarian response to gonadotropin and phorbol ester during the annual reproductive cycle. The effects of photothermal manipulation on perch gonadal steroidogenesis and its regulation have also been examined. Initially, all females kept indoors were exposed to the same water temperature and photoperiod. By the end of August, following the first sampling, fish were submitted to different photothermal regimes. Group A was maintained under photothermal conditions characteristic for southern Ohio. Group B was submitted to a condensed light/temperature regime designed to accelerate physiological changes that depend on photothermal stimuli. In group A, basal in vitro production of ovarian estradiol (E2) was the highest in October and November, and hCG significantly stimulated E2 secretion during the entire period of vitellogenesis (October-January). In this group, the highest production of basal testosterone (T) was observed before spawning. hCG-stimulated production of T was highest at the beginning of vitellogenesis. Gonadotropin stimulated T production before spawning, a time when gonadotropin was unable to stimulate E2 production. Phorbol ester (PDBu) stimulated E2 and T production during vitellogenesis at the same time points as hCG did (E2: December, January; T: December). hCG-stimulated T production was not mimicked by PDBu in April. Condensing of the photothermal cycle resulted in diminished ovarian production of E2 during vitellogenesis. Moreover, the fish submitted to a condensed photothermal cycle demonstrated augmented T production during the postvitellogenic stage of ovarian development. Ovaries of group B did not respond to PDBu. Generally, the seasonal fluctuations in ovarian capacity to produce E2 and T as well as in gonadal responsiveness to gonadotropin observed in female yellow perch illustrate the dynamic nature of ovarian endocrine function. The lack of response to gonadotropin with regard to E2 production prior to spawning is not due to insensitivity to gonadotropin, but rather due to some deficiency in steroidogenesis (e.g. reduced aromatase activity). It appears also that ovarian steroidogenesis and its regulation are dependent on annual changes of photothermal conditions.     

An in vitro model of gonad differentiation in the chicken. Estradiol-induced sex-inversion results in the occurrence of serological H-Y antigen

Dissociated cells from the gonads and mesonephros of 8-day-old chicken embryos were reorganized in rotation culture. The aggregates obtained from gonadal cells exhibited specific morphologic and histologic sex differences. In the presence of estradiol, aggregates from testicular cells showed characteristics similar to control ovarian aggregates, while in ovarian aggregates under estradiol treatment the female organization became more pronounced. Determination of serological H-Y antigen revealed that male aggregates of gonads and mesonephros were negative for H-Y and those of female embryos were positive for H-Y. Administration of estradiol did not change the H-Y findings in female aggregates. In contrast, in the male, gonadal cultures became H-Y positive while mesonephros cultures remained negative. It is assumed that estradiol induces the occurrence of H-Y antigen in the gonads.     

Increasing age influences uterine integrity, but not ovarian function or oocyte quality, in the cheetah (Acinonyx jubatus)

Although the cheetah (Acinonyx jubatus) routinely lives for more than 12 yr in ex situ collections, females older than 8 yr reproduce infrequently. We tested the hypothesis that reproduction is compromised in older female cheetahs due to a combination of disrupted gonadal, oocyte, and uterine function/integrity. Specifically, we assessed 1) ovarian response to gonadotropins; 2) oocyte meiotic, fertilization, and developmental competence; and 3) uterine morphology in three age classes of cheetahs (young, 2-5 yr, n = 17; prime, 6-8 yr, n = 8; older, 9-15 yr, n = 9). Ovarian activity was stimulated with a combination of equine chorionic gonadotropin and human chorionic gonadotropin (hCG), and fecal samples were collected for 45 days before gonadotropin treatment and for 30 days after oocyte recovery by laparoscopy. Twenty-six to thirty hours post-hCG, uterine morphology was examined by ultrasound, ovarian follicular size determined by laparoscopy, and aspirated oocytes assessed for nuclear status or inseminated in vitro. Although no influence of age on fecal hormone concentrations or gross uterine morphology was found (P > 0.05), older females produced fewer (P < 0.05) total antral follicles and oocytes compared to younger counterparts. Regardless of donor age, oocytes had equivalent (P > 0.05) nuclear status and ability to reach metaphase II and fertilize in vitro. A histological assessment of voucher specimens revealed an age-related influence on uterine tissue integrity, with more than 87% and more than 56% of older females experiencing endometrial hyperplasia and severe pathologies, respectively. Our collective findings reveal that lower reproductive success in older cheetahs appears to be minimally influenced by ovarian and gamete aging and subsequent dysfunction. Rather, ovaries from older females are responsive to gonadotropins, produce normative estradiol/progestogen concentrations, and develop follicles containing oocytes with the capacity to mature and be fertilized. A more likely cause of reduced fertility may be the high prevalence of uterine endometrial hyperplasia and related pathologies. The discovery that a significant proportion of oocytes from older females have developmental capacity in vitro suggests that in vitro fertilization and embryo transfer may be useful for "rescuing" the genome of older, nonreproductive cheetahs.     

The endocrine function of rat gonads with reduced number of germ cells following busulphan treatment

The endocrine function of rat gonads with an experimentally reduced number of germ cells was examined to analyse the effect of germ cells on the surrounding somatic endocrine cells. Pregnant Wistar rats received a single i.v. injection of 10 mg/kg B.W. of Busulphan on day 15 of gestation to prevent fetal primordial germ cells from starting mitotic division. The gonadal growth and the number of germ cells in Busulphan-treated rats (B-rats) were severely arrested. Almost a normal testicular structure was formed in the males, while few follicular structures were formed in the females, suggesting that the presence of oocytes in the fetal ovary is a prerequisite to the formation of the follicle. The meiotic division of spermatogonia in B-rats, which started on day 20 as in controls, stopped before the completion of spermatogenesis, and germ cells disappeared by day 50. The remaining germ cells and the associated follicles in female B-rats also disappeared by day 60 after repeating irregular estrous cycles for approximately 1 month. Thereafter the ovary consisted of fibroblasts and morphologically interstitial-like cells. Vaginal opening occurred in B-rats on day 28-30, a-week earlier than in controls. Changes in serum GTH after ovariectomy and the estradiol treatment suggested the maturation of the negative feedback sensitivity to estradiol in this period, and besides, earlier estradiol production with less dependency on gonadotropin. The vaginal epithelium of B-rats was cornified continuously after day 60. The ovarian cells in this period did not luteinize either morphologically or functionally in response to an ovulatory dose of hCG. During the same period, the conversion rate from progesterone to estradiol in ovarian homogenates of B-rats was considerably higher than those of controls at any stage of the estrous cycle. High content of estradiol was detected in the testes of B-rats at any age. In male B-rats, both LH and FSH levels in serum were higher than controls. The serum testosterone concentration in B-rats was lower than the normal, while the testicular testosterone content was greater. In conclusion, with a decreased number of germ cells, the rat gonads of both sexes secrete estradiol very efficiently.     

Regulation of infant and developing rat testicular gonadotropin and prolactin receptors and steroidogenesis by treatments with human chorionic gonadotropin, gonadotropin-releasing hormone analogs, bromocriptine, prolactin, and estrogen

Infant (5-day-old) male rats were treated with hormonal regimens to alter their exposure to gonadotropins, prolactin (Prl), and estrogen, and the response of testicular endocrine functions was measured. Human chorionic gonadotropin (hCG) or a potent gonadotropin-releasing hormone agonist analog (GnRH-A) resulted in a short-lived decrease of testicular receptors (R) for luteinizing hormone (LH), but no deleterious effects were found on testicular capacity to produce testosterone (T), which is a typical response of the adult testis. Only GnRH-A, through probable direct testicular action, induced a relative blockade of C21 steroid side-chain cleavage that was observed in vitro upon hCG stimulation. Human chorionic gonadotropin treatment, but not GnRH-A treatment, increased testicular Prl-R. GnRH antagonist analog (GnRH-Ant) treatment did not affect testicular LH-R, but decreased Prl-R and testicular T production. Decrease of serum Prl by bromocriptine had no effect on testicular LH-R or Prl-R, but slightly decreased T production in vitro. Ovine Prl increased binding sites for LH/hCG. The postnatal rats were insensitive to negative effects of diethylstilbestrol when monitored by testis weight, T, and LH-R. In conclusion, the responses to changes in the hormonal environment differed greatly between infant and adult testes. Mainly positive effects of elevated gonadotropin and Prl levels were seen on infant rat Leydig cell functions. Likewise, decreased tropic hormone levels, and exposure to estrogen, were ineffective in bringing about the inhibitory actions seen in the adult.     

Non-invasive faecal steroid monitoring of ovarian and adrenal activity in farmed blue fox (Alopex lagopus) females during late pregnancy, parturition and lactation onset

In the semi-domesticated blue fox, handling stress may influence reproductive performance and increase perinatal pup loss. Ovarian and adrenal steroids were analysed in faecal samples collected from mid-gestation through the first week of lactation in 40 female blue foxes to characterize hormone patterns during this important reproductive period. Daily faecal samples were collected from 40 foxes during 30 pregnancies, one late abortion and nine bred-matched non-pregnancies. Mean concentrations of faecal progestagens over the 10 days before birth were significantly higher in pregnant compared to non-pregnant females (51+/-1.50 microg/g versus 36+/-3.72 microg/g, respectively; P < 0.01). From 10 to 3 days before whelping, total faecal oestrogen concentrations also were higher (P < 0.01) in pregnant (1082+/-41.69 ng/g) than non-pregnant (628+/-72.43 ng/g) foxes, before declining to non-pregnant values (402+/-24.88 ng/g) after parturition. Overall mean faecal corticoid concentrations from 3 to 20 days before whelping differed between pregnant and non-pregnant foxes (128+/-3.11 ng/g versus 103+/-5.86 ng/g, respectively; P < 0.01). Furthermore, in pregnant foxes, corticoid excretion increased further from 2 days before to 3 days after whelping (216+/-13.71 ng/g; P < 0.01). Thereafter, corticoid concentrations were similar between pregnant and non-pregnant females (P > 0.05). In sum, the faecal steroid hormone patterns for oestrogens and progestagens were similar to those previously obtained by analyses of fox serum hormones, with both steroids being higher in pregnant than non-pregnant foxes at the end of gestation. The elevation in corticoid concentrations in pregnant females suggests that adrenal activation is involved in the initiation of parturition in the blue fox. Thus, faecal steroid analyses can be used to monitor ovarian activity during pregnancy and pseudopregnancy in farmed blue fox females.     

Increased rat testicular heme oxygenase activity associated with depressed microsomal heme and cytochrome P-450 levels after repeated administration of human chorionic gonadotropin

Repeated administration of human chorionic gonadotropin to rats results in a maximal depression of testicular microsomal heme and cytochrome P-450 levels at 24 h, followed by increases that plateau at pretreatment levels by day six. Associated with the depressed levels of microsomal heme and cytochrome P-450 is an increase of testicular microsomal heme oxygenase activity at 12-24 h. Testicular mitochondrial delta-aminolevulinic acid synthase activity was increased at 24 h, and remained elevated throughout the 9-day treatment period. Pretreatment with 1,4,6-androstatrien-3,17-dione, an aromatase inhibitor, failed to prevent the depression of testicular microsomal heme or cytochrome P-450 or increased heme oxygenase activity caused by repeated administration of human chorionic gonadotropin, and administration of estradiol benzoate failed to alter testicular microsomal heme oxygenase activity suggesting that these parameters were not related to altered testicular estrogen content caused by increased aromatase activity. These results suggest that increased testicular heme oxygenase activity is associated with decreased microsomal heme and cytochrome P-450 content during human chorionic gonadotropin-induced desensitization.     

The testosterone level in the testes of silver foxes during prenatal development]

The level of testosterone in serum and testes of the silver fox fetuses on days 31, 35, 40, 45, and 50 of gestation was determined using radioimmunoassay. In testes, testosterone was first detected at day 31; then its level gradually increased. Serum testosterone was detected only at day 40. Subsequent increase in its concentration was insignificant. Human chorionic gonadotropin stimulated testicular testosterone production in vitro beginning from day 40. We suggest that in the silver fox, testes are capable of responding to gonadotropins already by the day 40 of prenatal development but the formation of pituitary-gonadal axis proceeds until the end of embryogenesis.     

The effect of prenatal stress on the carboxypeptidase H activity in the hypothalamo-pituitary-adrenal-gonadal system of the rat

Effects of dams stress on the carboxypeptidase H: the neuropeptide exchange enzyme in the hypothalamo-pituitary-adrenall-gonadal system, was studied in the litter of different age (0, 14, 28, 45, and 120 day after birth) was studied. The sex differences in dynamic of enzyme activity in intact animals are substantiated. The effect of prenatal stress on carboxypeptidase H activity dependes on age and sex of animals. Prenatal stress is altering during the age dynamics of enzyme activity. This in the pituitary gland and hypothalamus of prenatal stressed female rats was a control for the male rats. In the adrenal and gonadal gland of prenatal stressed males, this was a control for the female rats female. The role of carboxypeptidase H in pubescence and mechanisms of effect of prenatal stress on sex system functional are discussed.     

Genetics of the +p9 syndrome

Summary Four 46,XY siblings with congenital bilateral megalorchidia, macrogenitosomia, and severe mental deficiency were investigated. The testicular size was significantly larger than age-matched normal males. A normal hypothalamic-pituitary gonadotropin function was demonstrated by the finding of normal levels of luteinizing and follicle-stimulating hormones in blood samples drawn at frequent intervals and by normal responses to gonadotropin-releasing hormone and testosterone adminstration. A normal testicular function was shown by the finding of normal (a) plasma testosterone and estradiol levels, (b) gonadal response to human chorionic gonadotropin, (c) sperm analysis, and (d) morphology and cell architecture of the testes. Adrenal function was found to be within normal limits. These results demonstrated the existence of normofunctional testicular hyperplasia. The family studies suggested that this distinct congenital disorder is inherited as an X-linked recessive trait.Presented at the annual meeting of the American Society of Human Genetics, Baltimore, Md., October 8–11, 1975. Abstract published in: Amer. J. hum. Genet., 27/6, 23A (1975).     

Ontogeny of the gonadal receptor for luteinizing hormone in the pig

Homogenates of porcine ovaries and testes collected between 70 d post coitum and 42 d post partum were incubated with radiolabelled human chorionic gonadotropin (hCG) to determine the presence and relative amounts of luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptors. Specific binding of (125)I-hCG to ovaries and testes occurred at all stages of fetal and postnatal development. Ovarian tissue possessed relatively low affinity, high capacity LH/hCG binding sites that were most numerous at Day 80 of gestation and decreased thereafter. In contrast, high affinity, low capacity LH/hCG binding sites were found in the testes. In males, the total number of LH/hCG binding sites remained stable until near term and then increased with age, but the number of sites per gram of testicular tissue did not change (P>0.05). In summary, differential binding of LH/hCG in gonadal tissue occurred in male and female piglets during pre- and post-natal periods, and this binding reflected the known differential pattern of development of the male and female gonad.     

Milk intake in blue fox (Alopex lagopus) and silver fox (Vulpes vulpes) cubs in the early suckling period

Milk intake of fox cubs (2-16 days of age; body weight, 96-649 g) in ten blue fox litters and ten silver fox litters were measured by the water isotope dilution (WID) technique following a single intraperitoneal injection of tritiated water (3HHO). Litter size varied from four to 14 in blue foxes and from three to eight in silver foxes. Silver fox cubs had higher birth weights than blue foxes. Inter-species body weights and growth rates were apparently dependent on litter size and the dam's constitution. In both species growth rate increased with age and body weight (7-35 g per day). In the cubs, the biological half-life of body water turnover (BWT) rose from 1.5 days at 2-3 days of age to 2.5 days at 13-16 days of age, although a considerable scatter was seen. The mean daily milk intake of the cubs varied with body weight, from 31 to 193 g per day, whereas daily milk intake per unit of body mass remained stable at 30-35 g per 100 g body weight. The ratio of milk intake to body weight gain varied considerably among cubs, averaging 4.5 g/g during the 3-week experimental period. In suckling fox cubs, the calculated daily intake of metabolically energy (ME) corresponded fairly with the estimated energy requirements for growth and maintenance of the young. Finally, the applicability and the accuracy of the WID technique was evaluated in ten 3-week-old fox cubs, by tube-feeding with a milk replacer for 48 h, which documented that the daily rates of milk intake and water turnover can be accurately measured in suckling fox cubs by the WID technique following a single injection of 3HHO.     

Regulation of Oxidative Activity of Neutrophils by Chorionic Gonadotropin: The Role of Female Steroid Sex Hormones

We studied the effect of the main sex hormone, chorionic gonadotropin, on the production of the active forms of oxygen and nitrogen by human neutrophils and the control of its effects by female steroid sex hormones. Gonadotropin proved to inhibit both total production of oxygen metabolites and NO synthesis by the cells. Gonadotropin-dependent regulation of oxidative activity of the neutrophils is controlled by female steroid sex hormones. At the same time, progesterone completely or partially inhibits the suppressive effects of gonadotropin, while estradiol has antagonistic, sensibilizing, or permissive effect on gonadotropin depending on the dose, incubation conditions, and activation status of the cells.     

Regulation of an 8-kDa peptide involved in testosterone production by luteinizing hormone in rat Leydig cells.

Results of Western blot analysis carried out with an interstitial cell extract from male guinea pig and ovarian extract from immature female rats administered equine chorionic gonadotropin (eCG) provide supportive evidence to our earlier suggestion that an 8-kDa peptide is involved in acquisition of steroidogenic capacity by the rat Leydig cells. It was found that though the signal was observed in other tissues such as liver, kidney and lung which do not produce gonadal hormones, the peptide was modulated only by lutenizing hormone (LH) in the rat Leydig cells.