Hormonal control of arylhydrocarbon receptor (AhR) expression in the preimplantation rabbit uterus

We show that the arylhydrocarbon receptor (AhR) is localised in a distinct pattern in the preimplantation rabbit uterus. Employing immunohistochemistry, uteri from mature and immature rabbits were studied in different pregnancy stages during the preimplantation period (non-pregnant/estrous, pregnant and pseudopregnant) and following treatment with estradiol and progesterone, respectively. The staining patterns in pregnant and pseudopregnant rabbits were similar, indicating a primary maternal control of expression. A distinct change in AhR localization occurred in the endometrium of (1) non-pregnant compared with pregnant rabbits and (2) shortly prior to the anticipated time of implantation. Compared with non-pregnant uteri, the AhR was no longer restricted to a small cytoplasmic area apical to the nucleus but was found in the entire cytoplasm and in the nuclei of the endometrial epithelial cells in pregnant uteri. Prior to implantation, at day 6, stromal cells and the epithelium of the uterine glands, but no longer the luminal epithelial cells, were immunopositive. The estradiol-treated immature uteri showed a strong expression in the luminal epithelium and in some uterine glands, often with nuclear staining, while the estradiol-primed and progesterone-treated specimens had a similar staining pattern as mature preimplantation uteri. Present results demonstrate a distinct localization pattern of the AhR in the endometrium during the preimplantation period. Stage-specific changes in the expression pattern indicate control of AhR expression by maternal steroid hormones. Accepted: 9 July 2001     

Exploring the role of sex steroids through studies of receptor deficient mice

Decades of study have described a number roles fulfilled by the steroid hormones and their respective receptors in sexual differentiation and development, reproductive function and behavior, and more recently in the function and maintenance of non-reproductive organ systems, such as skeletal muscle, bone and coronary tissues. The biological effects of the steroid hormones are believed to be mediated in part by specific receptor proteins that demonstrated great specificity for their respective steroid ligands. Much of the experimental research of the functions of the sex steroid receptors has depended upon in vitro systems as well as in vivo methods that require surgical castration or the pharmacological administration of hormone antagonists. However, recently developed techniques that allow for manipulation of the mouse genome have been utilized to generate transgenic animals that lack functional estrogen or progesterone receptors. These transgenic animals, combined with the naturally existing Tfm mice which lack functional androgen receptor, now provide in vivo models for further study of the various actions of the sex steroids and their receptors. This review attempts to describe and compare the various phenotypes that result in each of these lines of mice, with emphasis on the development and function of the reproductive systems as well as reproductive behavior. Received: 19 June 1997 / Accepted: 25 September 1997     

Reproductive functions of corticotropin-releasing hormone. Research and potential clinical utility of antalarmins (CRH receptor type 1 antagonists)

BACKGROUND: The hypothalamic-pituitary-adrenal (HPA) axis exerts a complex, mostly inhibitory, effect on the female reproductive system. In addition, the principal regulator of this axis, the hypothalamic neuropeptide corticotropin-releasing hormone (CRH) and its receptors have been identified in most female reproductive tissues, including the ovary, uterus, and placenta. Furthermore, CRH is secreted in peripheral inflammatory sites where it exerts strong inflammatory actions. Antalarmins (CRH receptor type 1 antagonists) have been used to elucidate the roles of CRH in stress, inflammation and reproduction. METHOD OF STUDY: We review existing data on the effects of CRH in the female reproductive system. RESULTS: Ovarian CRH participates in female sex steroid production, follicular maturation, ovulation and luteolysis. Uterine CRH participates in decidualization, implantation, and early maternal tolerance. Placental CRH participates in the physiology of pregnancy and the onset of parturition. Circulating placental CRH is secreted mostly during the latter half of pregnancy and is responsible for the concurrently increasing physiologic hypercortisolism of this period. After labor and delivery, this hypercortisolism is ensued by a transient suppression of hypothalamic CRH secretion, which may explain the postpartum blues and depression and the increased autoimmune manifestations depression of period, the postpartum period. CONCLUSIONS: These data show that CRH is present in female reproductive tissues, and is regulating key reproductive functions with an inflammatory component, such as ovulation, luteolysis, implantation, and parturition.     

Effect of active and passive immunization of male and female rats with a recombinantly expressed bonnet monkey pituitary GnRH receptor fragment

Gonadotropin releasing hormone (GnRH) exerts its action by binding to the specific receptor which belongs to the family of G-protein coupled receptors that are characterized by the presence of seven transmembrane domains linked together by extracellular and intracellular loops. A fragment of the pituitary receptor of the bonnet monkey (Macaca radiata) corresponding to amino acids 164-266 was cloned and expressed in Escherichia coli. This was used to raise antibodies to the receptor in rabbits. Active and passive immunization studies in both male and female rats were carried out using, both the 'overexpressed' fragment, as well as antibodies raised to the receptor fragment. Both active, as well as passive immunization in the male rats brought about an agonistic effect in terms of increase in serum LH level, as well as increase in serum and testicular testosterone levels. However, in the female rats, active immunization with the receptor fragment did not have any effect on the gonadal steroid levels but had a selective effect on the uterine morphology.     

Luteinizing hormone/human chorionic gonadotropin receptors in myometrium and their role in uterine motility

The object of this paper is the existence of LH/hCG receptors in female uterine tissue. Specific and high-affinity binding sites for LH are present in the pig and rabbit uterus. Estradiol promoted the synthesis of LH receptors in porcine myometrium. Stimulation of LH receptors with hCG in estrogen-primed tissue had a quiescence effect on myometrial contractility in vitro. The physiological role of uterine LH receptors in maintenance of myometrial quiescence is discussed.     

Localization of 5-hydroxytryptamine-like immunoreactive cells and nerve fibers in the rat female reproductive system.

The presence of 5-hydroxytryptamine (5-HT)-like immunoreactivity (IR) was studied in the rat female reproductive system using polyclonal antibodies directed against 5-HT. Moreover, 5-HT levels in the ovary, oviduct, uterus, and cervix were measured by high-pressure liquid chromatography with electrochemical detection. The highest 5-HT concentrations were found in the oviduct, followed in descending order by the cervix, the ovary, and the uterus. Most 5-HT-like IR was observed in the cytoplasm of mast cells. These cells were found in the connective tissue around the fimbria, in the oviduct, in the uterus, and in the ovary. Mast cells are clustered in the proximity of the parenchymal blood vessels. Moreover, a few 5-HT-like nerve fibers were found distributed mainly perivascularily in the uterine cervix and in the uterine horns as well as in the oviduct. IR nerve fibers were rarely seen within the ovary. The present data provide direct evidence that 5-HT in the female reproductive system not only is associated with mast cells but is located in nerve fibre-like structures as well. The functional significance of this probable 5-HT-ergic innervation of the female reproductive tract discovered in the present study should be clarified in future investigations.     

Reproductive capacity of male laboratory rats

The purpose of this research was to investigate the reproductive capacity (i.e., the ability to successfully transfer an ejaculate and induce a progestational response in the female) of male laboratory rats (Rattus norvegicus). Males were individually mated to successive females in a single session for one ejaculatory series each. There was a marked decline in both the total number of sperm per ejaculate and the number of sperm transported to the uterus with repeated ejaculations. Dimensions of the copulatory plug of the male decreased over successive ejaculations, reflecting a diminished output of the secretions of the reproductive accessory glands. Male copulatory behavior changed over successive ejaculations. Actual fertility appeared somewhat diminished in late ejaculatory series. Such constraints on the reproductive capacity of the male may have implications for the mating strategy employed by wild males in the field.     

Protein restriction during puberty alters nutritional parameters and affects ovarian and uterine histomorphometry in adulthood in rats

In a large part of the population inefficient ingestion of proteins, whether for cultural, aesthetic or economic reasons, is a global concern. Low‐protein diets can cause severe functional complications, mainly during the development and maturation of organs and systems, including the female reproductive system. The present study investigated the effect of nutritional protein restriction during puberty on the oestrous cycle and expression of sex steroid receptors (AR, ERα e ERβ) in ovarian and uterine tissues of adult rats. Protein restriction promoted lower body weight gain, feed efficiency and higher caloric intake. There was an increase in the oestrus phase arrest without changing the total length of the oestrous cycle. The consumption of low‐protein diet also reduced the thickness of the uterine endometrium (uterine epithelium and endometrial stroma) in addition to increasing the number of primary and atretic follicles in the ovaries. Furthermore, the low‐protein diet reduced the levels of androgen receptor (AR) and increased the oestrogen receptor β (ERβ) in the ovary, while no significant changes were observed in the uterus. Our study reinforces the importance of adequate protein intake during puberty, since physiological changes in this developmental period interfere with the histomorphometry of the ovaries and uteri, possibly resulting in impaired folliculogenesis and fertility in the reproductive period.     

Longer term progesterone treatment induces changes of GABAA receptor levels in forebrain sites in the female hamster: quantitative autoradiography study

Summary Quantitative receptor autoradiography was applied to evaluate the effects of one and three injections of 1 mg progesterone (P) on ³H muscimol binding levels in the different forebrain areas of the female hamster. The overall effect of P resulted in substantial increases in ³H muscimol binding in brain areas containing gonadal steroid receptors: medial preoptic area and ventromedial hypothalamic nucleus as well as in bed nucleus stria terminalis and subiculum. Similarly, the caudate putamen, a region where gonadal steroid receptors are not abundant, also showed substantial increases of ³H muscimol binding receptor levels. Moreover, female hamsters treated with P for 3 days presented altered ³H muscimol binding levels in the amygdala and thalamic nucleus that were, in some cases, not produced by one dose of P. P treatment also decreased GABA_A binding in two areas of the thalamus. These results are consistent with the proposal that P may alter GABAergic inhibitory activity via changes in the levels of GABA_A receptors in certain forebrain areas in the female hamster, changes which may be linked to the mediation of anxiolytic effects and to the inhibition of aggressive behavior. These data also suggest that P treatment increases the binding of high affinity GABA receptors in some forebrain sites and may be responsible for maintenance of the anxiolytic effects.     

Localization of 5-hydroxytryptamine-like immunoreactive cells and nerve fibers in the rat female reproductive system

The presence of 5-hydroxytryptamine (5-HT)-like immunoreactivity (IR) was studied in the rat female reproductive system using polyclonal antibodies directed against 5-HT. Moreover, 5-HT levels in the ovary, Oviduct, uterus, and cervix were measured by high-pressure liquid chromatography with electrochemical detection. The highest 5-HT concentrations were found in the oviduct, followed in descending order by the cervix, the ovary, and the uterus. Most 5-HT-like IR was observed in the cytoplasm of mast cells. These cells were found in the connective tissue around the fimbria, in the oviduct, in the uterus, and in the ovary. Mast cells are clustered in the proximity of the parenchymal blood vessels. Moreover, a few 5-HT-like nerve fibers were found distributed mainly perivascularily in the uterine cervix and in the uterine horns as well as in the oviduct. IR nerve fibers were rarely seen within the ovary. The present data provide direct evidence that 5-HT in the female reproductive system not only is associated with mast cells but is located in nerve fibre-like structures as well. The functional significance of this probable 5-HT-ergic innervation of the female reproductive tract discovered in the present study should be clarified in future investigations. © 1992 Wiley-Liss, Inc.     

Mutant forms of tumour necrosis factor receptor I that occur in TNF-receptor-associated periodic syndrome retain signalling functions but show abnormal behaviour

Tumour necrosis factor (TNF)-receptor-associated periodic syndrome (TRAPS) is a hereditary autoinflammatory disorder involving autosomal-dominant missense mutations in TNF receptor superfamily 1A (TNFRSF1A) ectodomains. To elucidate the molecular effects of TRAPS-related mutations, we transfected HEK-293 cells to produce lines stably expressing high levels of either wild-type (WT) or single mutant recombinant forms of TNFRSF1A. Mutants with single amino acid substitutions in the first cysteine-rich domain (CRD1) were produced both as full-length receptor proteins and as truncated forms lacking the cytoplasmic signalling domain (deltasig). High-level expression of either WT or mutant full-length TNFRSF1A spontaneously induced apoptosis and interleukin-8 production, indicating that the mutations in CRD1 did not abrogate signalling. Consistent with this, WT and mutant full-length TNFRSF1A formed cytoplasmic aggregates that co-localized with ubiquitin and chaperones, and with the signal transducer TRADD, but not with the inhibitor, silencer of death domain (SODD). Furthermore, as expected, WT and mutant deltasig forms of TNFRSF1A did not induce apoptosis or interleukin-8 production. However, whereas the WT full-length TNFRSF1A was expressed both in the cytoplasm and on the cell surface, the mutant receptors showed strong cytoplasmic expression but reduced cell-surface expression. The WT and mutant deltasig forms of TNFRSF1A were all expressed at the cell surface, but a proportion of the mutant receptors were also retained in the cytoplasm and co-localized with BiP. Furthermore, the mutant forms of surface-expressed deltasig TNFRSF1A were defective in binding TNF-alpha. We conclude that TRAPS-related CRD1 mutants of TNFRSF1A possess signalling properties associated with the cytoplasmic death domain, but other behavioural features of the mutant receptors are abnormal, including intracellular trafficking and TNF binding.     

Mucin expression and function in the female reproductive tract

Reproductive tract epithelia are characterized by the presence of a thick, apical glycocalyx. This glycoprotein coat is drastically reduced in the uterus of many species during the time of embryo implantation. Recent studies indicate that mucin glycoproteins constitute a large proportion of the apical glycocalyx. One of these mucins, Muc-1, has particularly important functions at the luminal surface of the uterus and other female reproductive tract tissues. Muc-1 appears to play a dominant role in maintaining a functionally non-receptive uterine surface with regard to blastocyst attachment. Conversion to a receptive uterine state is brought about by the concerted actions of ovarian steroid hormones that in several species also strongly modulate Muc-1 protein and mRNA expression. Muc-1 also appears to serve a general function in protecting reproductive tract mucosa since Muc-1 null mice are particularly prone to bacterial infection. Collectively, these studies indicate that mucins, including Muc-1, play important barrier roles in reproductive processes and protection from bacterial pathogenesis in the female reproductive tract.     

Effects of cholecystokinin on male copulatory behavior and lordosis behavior in male rats

Because the distribution of cholecystokinin octapeptide (CCK-8) within the hypothalamus and limbic system overlaps with steroid concentrating regions, and because these areas are involved in the regulation of reproductive behaviors, we examined the effects of exogenous CCK-8 on male copulatory behavior and lordosis behavior in the male rat. Peripheral administration of a dose of CCK-8 that altered lordosis behavior in females (3 micrograms/kg, intraperitoneal) was ineffective in altering male copulatory behavior in males, either before or after gonadectomy, and was also ineffective in altering lordosis behavior after estrogen priming. In a separate experiment, CCK-8 injected into the lateral ventricle also did not affect male copulatory behavior, but lordosis behavior was increased dramatically after gonadectomy and estrogen priming. Although these results do not answer the question whether CCK-8 is acting to inhibit a neural system that normally suppresses lordosis behavior or is acting to stimulate a facilitatory circuit, these results do indicate the existence of an estrogen sensitive neural substrate in males on which CCK can act to facilitate lordosis behavior.     

Treatment with mifepristone (RU486) and oestradiol facilitates the development of genital septic disease after copulation in female rats.

Ovariectomized female rats were treated with oestradiol and the progesterone-antagonist mifepristone. They allowed males to copulate to a similar extent as ovariectomized rats treated with oestradiol alone. The additional treatment with mifepristone, however, resulted in uterine infections following copulation, together with the prolonged presence of copulatory plugs in the uterine lumen. On several occasions rats became severely ill during the days after copulatory tests, occasionally with lethal consequences. Microscopic examination of the cervix and uterus 1 day after a copulation test in rats treated with oestradiol plus mifepristone, showed that copulatory plugs passed through the cervical canals directly into the uterine lumen. Bacterial infections and local destruction of the uterine epithelium were found in all rats examined. These features were not found in rats treated with oestradiol alone. Actions of mifepristone on the cervix of oestradiol treated rats are likely to play a key role in the passage of copulatory plugs and, thereafter, the development of uterine sepsis.     

Immunohistochemical detection of the estrogen receptor‐α and progesterone receptor in the canine pregnant uterus and placental labyrinth

The presence of hormone receptors is as important as the amount of hormone to predict hormone action. Therefore, the presence of estrogen receptors of the alpha subtype (ER‐α) and progesterone receptors (PR) was evaluated in six pregnant uteri including the placenta and in three postpartum uteri of dogs. This preliminary study is part of our immunohistochemical research project on steroid hormone receptor distribution in the canine female genital tract. Specific staining for ER‐α or PR was found only in cell nuclei. Staining for ER‐α was rare in the various cell types of pregnant and postpartum uteri. Staining for PR was absent or weak in epithelial cells. Moderate staining for PR was observed in endometrial stromal cells and myometrial smooth muscle cells, two cell types playing an important role in the maintenance of pregnancy. Stromal cells stained more frequently positive for ER‐α and PR than epithelial cells, indicating that both hormones may act on epithelial cells indirectly via stromal cells. In the placental labyrinth, fetal cells showed no evidence of ER‐α or PR. In contrast, both receptors were present in maternal mesenchymal cells that were located around the basement membrane of the maternal blood vessels. These cells showed signs of decidualization. No difference in PR distribution was seen between pregnant and postpartum uterine tissue, suggesting that during parturition the decrease in serum progesterone levels and the concomitant increase in the estrogen/progesterone ratio are probably more important than the decline in receptor availability. Anat Rec 260:42–50, 2000. © 2000 Wiley‐Liss, Inc.     

Evidence for direct regulation of epidermal growth factor receptors by steroid hormones in human endometrial cells.

Recent evidence suggests that epidermal growth factor (EGF) may serve as a paracrine and/or autocrine mediator of oestrogen action in uterine growth. In this study, the effects of various steroid hormones on EGF receptors in primary cultures of human endometrial cells were examined. Human endometrial cells exhibited a single class of high-affinity binding sites for EGF (Kd: 0.14 nM) with approximately 3600 receptors/cell. The addition of progesterone increased the EGF binding without affecting the Kd value. Cortisol also increased EGF binding and acted additively with a relatively low concentration of progesterone (10(-10) M). Oestradiol alone had no effect on EGF binding. However, oestradiol in combination with progesterone and cortisol further increased EGF binding. These results present evidence for the direct regulation of EGF receptors by steroid hormones in human endometrial cells, and raise the possibility that steroid hormones may act on these cells in part by modulating EGF receptors.     

The posteromedial cortical amygdala regulates copulatory behavior, but not sexual odor preference, in the male Syrian hamster (Mesocricetus auratus)

In rodent species, the expression of reproductive behavior relies heavily on the perception of social odors, as well as the presence of circulating steroid hormones. In the Syrian hamster, chemosensory and hormonal cues are processed within an interconnected network of ventral forebrain nuclei that regulates many aspects of social behavior. Within this network, the posteromedial cortical amygdala (PMCo) receives direct projections from the accessory olfactory bulbs and contains a dense population of steroid receptor-containing neurons. Consequently, the PMCo may be important for generating odor-guided aspects of reproductive behavior, yet little is known regarding the role of this nucleus in regulating these behaviors. Thus, the present study tested male hamsters with site-specific electrolytic lesions of the PMCo for their (a) sexual odor preference in a Y-maze apparatus, (b) sexual odor discrimination in a habituation-dishabituation task, and (c) copulatory behavior when paired with a sexually receptive female. PMCo-lesioned males preferred to investigate female odors over male odors and were able to discriminate between these odor sources. However, PMCo lesions were associated with several alterations in the male copulatory pattern. First, PMCo-lesioned males displayed increased investigation of the female's non-anogenital region, suggesting that the PMCo may be involved in directing appropriate chemosensory investigation during mating. Second, PMCo lesions altered the temporal pattern of the mating sequence, as PMCo-lesioned males took longer than Sham-lesioned males to reach sexual satiety, as indicated by the delayed expression of long intromissions. This delayed onset of satiety was associated with an increased number of ejaculations compared with Sham-lesioned males. Importantly, these data provide the first direct evidence for a functional role of the PMCo in regulating male reproductive behavior.     

Effect of flutamide (an antiandrogen) and diethylstilbestrol on the reproductive behavior of Japanese quail

Three experiments were conducted in order to investigate the role of brain androgen and estrogen receptors in sex hormone activated male reproductive behavior in Japanese quail. In Experiment 1, castrated male quail were injected with oil, testosterone propionate (TP), flutamide (FLUT), an androgen antagonist, or TP+FLUT. Males given TP+FLUT, compared with birds receiving TP alone, strutted much less and had smaller proctodeal (foam) glands. Copulation was reduced by FLUT only on the last test day and only on one measure (number of head grabs + mounts). These results suggest that binding of testosterone or one of its metabolites to an androgen receptor is part of the mechanism of TP activated strutting, and therefore that central nervous system androgen receptors are involved in a male reproductive behavior pattern. In Experiment 2, castrated male quail were injected with oil, with 50 micrograms estradiol benzoate (EB), or with 25, 50 or 100 micrograms diethylstilbestrol (DES), a synthetic estrogen that does not bind to androgen receptors. EB but not DES activated copulation to a significant extent. In Experiment 3 male and female quail with photically regressed gonads were given intraperitoneal Silastic implants of DES, estradiol (E) or cholesterol. DES was highly effective at activating male-typical copulation in males and receptivity in both sexes. Thus hormonal interaction with estrogen receptors alone is sufficient for the activation of male-typical as well as female-typical copulatory behavior in this species.