All scientific investigations begin with distinct objectives: first is the hypothesis upon which studies are undertaken to disprove, and second is the overall aim of obtaining further information, from which future and more precise hypotheses may be drawn Studies focusing on the generation and use of gene-targeted animal models also apply these goals and may be loosely categorized into sequential phases that become apparent as the use of the model progresses Initial studies of knockout models often focus on the plausibility of the model based on prior knowledge and whether the generation of an animal lacking the particular gene will prove lethal or not Upon the successful generation of a knockout, confirmatory studies are undertaken to corroborate previously established hypotheses of the function of the disrupted gene product As these studies continue, observations of unpredicted phenotypes or, more likely, the lack of a phenotype that was expected based on models put forth from past investigations are noted Often the surprising phenotype is due to the loss of a gene product that is downstream from the functions of the disrupted gene, whereas the lack of an expected phenotype may be due to compensatory roles filled by alternate mechanisms As the descriptive studies of the knockout continue, use of the model is often shifted to the role as a unique research reagent, to be used in studies that 1) were not previously possible in a wild-type model; 2) aimed at finding related proteins or pathways whose existence or functions were previously masked; or 3) the subsequent effects of the gene disruption on related physiological and biochemical systems The alpha ERKO mice continue to satisfy the confirmatory role of a knockout quite well As summarized in Table 4, the phenotypes observed in the alpha ERKO due to estrogen insensitivity have definitively illustrated several roles that were previously believed to be dependent on functional ER alpha, including 1) the proliferative and differentiative actions critical to the function of the adult female reproductive tract and mammary gland; 2) as an obligatory component in growth factor signaling in the uterus and mammary gland; 3) as the principal steroid involved in negative regulation of gonadotropin gene transcription and LH levels in the hypothalamic-pituitary axis; 4) as a positive regulator of PR expression in several tissues; 5) in the positive regulation of PRL synthesis and secretion from the pituitary; 6) as a promotional factor in oncogene-induced mammary neoplasia; and 7) as a crucial component in the differentiation and activation of several behaviors in both the female and male The list of unpredictable phenotypes in the alpha ERKO must begin with the observation that generation of an animal lacking a functional ER alpha gene was successful and produced animals of both sexes that exhibit a life span comparable to wild-type The successful generation of beta ERKO mice suggests that this receptor is also not essential to survival and was most likely not a compensatory factor in the survival of the alpha ERKO In support of this is our recent successful generation of double knockout, or alpha beta ERKO mice of both sexes The precise defects in certain components of male reproduction, including the production of abnormal sperm and the loss of intromission and ejaculatory responses that were observed in the alpha ERKO, were quite surprising In turn, certain estrogen pathways in the alpha ERKO female appear intact or unaffected, such as the ability of the uterus to successfully exhibit a progesterone-induced decidualization response, and the possible maintenance of an LH surge system in the hypothalamus [ABSTRACT TRUNCATED]

Estrogen receptor null mice: what have we learned and where will they lead us?

Estrogens influence the differentiation and maintenance of reproductive tissues and affect lipid metabolism and bone remodeling. Two estrogen receptors (ERs) have been identified to date, ERα and ERβ. We previously generated and studied knockout mice lacking estrogen receptor α and reported severe reproductive and behavioral phenotypes including complete infertility of both male and female mice and absence of breast tissue development. Here we describe the generation of mice lacking estrogen receptor β (ERβ −/−) by insertion of a neomycin resistance gene into exon 3 of the coding gene by using homologous recombination in embryonic stem cells. Mice lacking this receptor develop normally and are indistinguishable grossly and histologically as young adults from their littermates. RNA analysis and immunocytochemistry show that tissues from ERβ −/− mice lack normal ERβ RNA and protein. Breeding experiments with young, sexually mature females show that they are fertile and exhibit normal sexual behavior, but have fewer and smaller litters than wild-type mice. Superovulation experiments indicate that this reduction in fertility is the result of reduced ovarian efficiency. The mutant females have normal breast development and lactate normally. Young, sexually mature male mice show no overt abnormalities and reproduce normally. Older mutant males display signs of prostate and bladder hyperplasia. Our results indicate that ERβ is essential for normal ovulation efficiency but is not essential for female or male sexual differentiation, fertility, or lactation. Future experiments are required to determine the role of ERβ in bone and cardiovascular homeostasis.

https://www.pnas.org/content/pnas/95/26/15677.full.pdf

Generation and reproductive phenotypes of mice lacking estrogen receptor β

Significant advances have taken place in our knowledge of the enzymes involved in steroid hormone biosynthesis since the last comprehensive review in 1988. Major developments include the cloning, identification, and characterization of multiple isoforms of 3β-hydroxysteroid dehydrogenase, which play a critical role in the biosynthesis of all steroid hormones and 17β-hydroxysteroid dehydrogenase where specific isoforms are essential for the final step in active steroid hormone biosynthesis. Advances have taken place in our understanding of the unique manner that determines tissue-specific expression of P450aromatase through the utilization of alternative promoters. In recent years, evidence has been obtained for the expression of steroidogenic enzymes in the nervous system and in cardiac tissue, indicating that these tissues may be involved in the biosynthesis of steroid hormones acting in an autocrine or paracrine manner. This review presents a detailed description of the enzymes involved in the biosynthe...

https://www.researchgate.net/profile/Dale_Buck_Hales/publication/8144948_Overview_of_steroidogenic_enzymes_in_the_pathway_from_cholesterol_to_active_steroid_hormones/links/02e7e5249ac2529059000000.pdf

Overview of Steroidogenic Enzymes in the Pathway from Cholesterol to Active Steroid Hormones

This report is an overview of the current state of the science relative to environmental endocrine disruption in humans, laboratory testing, and wildlife species. Background information is presented on the field of endocrinology, the nature of hormones, and potential sites for endocrine disruption, with specific examples of chemicals affecting these sites. An attempt is made to present objectively the issue of endocrine disruption, consider working hypotheses, offer opposing viewpoints, analyze the available information, and provide a reasonable assessment of the problem. Emphasis is placed on disruption of central nervous system--pituitary integration of hormonal and sexual behavioral activity, female and male reproductive system development and function, and thyroid function. In addition, the potential role of environmental endocrine disruption in the induction of breast, testicular, and prostate cancers, as well as endometriosis, is evaluated. The interrelationship of the endocrine and immune system is documented. With respect to endocrine-related ecological effects, specific case examples from the peer-reviewed literature of marine invertebrates and representatives of the five classes of vertebrates are presented and discussed. The report identifies some data gaps in our understanding of the environmental endocrine disruption issue and recommends a few research needs. Finally, the report states the U.S. Environmental Protection Agency Science Policy Council's interim position on endocrine disruption and lists some of the ongoing activities to deal with this matter.

Environmental endocrine disruption: an effects assessment and analysis.

Two major functions of the mammalian ovary are the production of germ cells (oocytes), which allow continuation of the species, and the generation of bioactive molecules, primarily steroids (mainly estrogens and progestins) and peptide growth factors, which are critical for ovarian function, regulation of the hypothalamic-pituitary-ovarian axis, and development of secondary sex characteristics. The female germline is created during embryogenesis when the precursors of primordial germ cells differentiate from somatic lineages of the embryo and take a unique route to reach the urogenital ridge. This undifferentiated gonad will differentiate along a female pathway, and the newly formed oocytes will proliferate and subsequently enter meiosis. At this point, the oocyte has two alternative fates: die, a common destiny of millions of oocytes, or be fertilized, a fate of at most approximately 100 oocytes, depending on the species. At every step from germline development and ovary formation to oogenesis and ovaria...

/pdf/the-mammalian-ovary-from-genesis-to-revelation-2e2qokz0h8.pdf

The mammalian ovary from genesis to revelation

The effects of estradiol, FSH and LH on ovarian follicular development and granulosa cell differentiation were examined in the immature rat hypophysectomized on day 24 of age. Administration of estradiol to hypophysectomized rats for 4 days stimulated the growth of large preantral follicles with a concomitant 1.5-fold increase in FSH receptor content and a 4-fold decrease in LH receptor content in the granulosa cells. When highly purified hFSH was administered alone, receptor content for FSH increased progressively for 4 days while receptor for LH remained essentially unchanged. However, when rats were pretreated with estradiol, the response of follicles to FSH was markedly enhanced as indicated by the appearance of large, antral follicles and elevated receptor content for both FSH and LH. Receptor content for FSH increased markedly in response to hFSH following only one day of estradiol pretreatment, while receptor content for LH increased most rapidly in response to hFSH after 3 days of estradiol pretreatment. LH administered to rats possessing large preovulatory follicles caused luteinization of granulosa cells and a marked decline in receptor content for both gonadotropins within 24 h. Receptor content remained low even 48 h after LH administration when granulosa cells were fully luteinized. These results indicated that follicular development and granulosa cell differentiation are dependent on steroid-protein hormone regulation of hormone specific receptors.

Ovarian follicular development in the rat: hormone receptor regulation by estradiol follicle stimulating hormone and luteinizing hormone.

Hormonal regulation of ovarian cellular proliferation

The following study was undertaken to compare the content of aromatase cytochrome P450 (P450arom) mRNA with the content of the enzyme in rat ovarian tissues and to relate these changes with estradiol biosynthesis by follicles and corpora lutea isolated throughout pregnancy. A deoxyoligonucleotide (62 mer) probe derived from an amino acid sequence of purified human placental P450arom was used to screen a rat granulosa cell lambda gt11 cDNA expression library. Seven cDNA clones, ranging in size from 0.6-2.0 kilobases (kb), were identified and plaque purified. In vitro translation using mRNA that had been selected by hybridization to a 1.2-kb rat P450arom cDNA insert yielded an 35S-labeled translation product that bound antihuman aromatase immunoglobulin and comigrated with purified human placental aromatase on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, thus verifying that the clones do encode for P450arom. Using the 1.2-kb cDNA insert as a radiolabeled probe, the hormonal regulation, tissue distribution, content, and size of mRNA for P450arom were analyzed. Filter hybridization assays demonstrated that P450arom mRNA was low in small antral (SA) follicles, increased 16-fold in preovulatory (PO) follicles, and reached a peak in granulosa cells within 1 h after an ovulatory dose of hCG. In the corpus luteum of pregnancy, P450arom mRNA content was low on day 4, and increased 3-fold on days 7-11 and 10-fold on days 15-19 of gestation. P450arom mRNA then decreased on days 21 and 23, the day of parturition. Northern analyses of RNA from PO follicles and corpora lutea revealed three bands of P450arom mRNA that were 3.3, 2.6, and 1.9 kb in size. Immunoblots of soluble cell extracts of SA, PO, and luteinizing (PO plus hCG) follicles and corpora lutea of pregnancy demonstrated that aromatase enzyme was low in SA follicles, increased 1.5- to 3-fold in PO follicles, and decreased within 3-5 h after an ovulatory dose of hCG. Changes in the content of P450arom enzyme in luteal cells during pregnancy exhibited a pattern similar to that observed for P450arom mRNA. In contrast, changes in estradiol biosynthesis by follicles and corpora lutea were not directly related to the contents of P450arom mRNA and enzyme. For example, although corpora lutea isolated on days 15-21 of gestation contain the highest amount of P450arom mRNA and enzyme, these tissues did not produce the most estradiol when incubated for 5 h at 37 C in the presence of aromatizable androgen substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

Hormonal regulation, tissue distribution, and content of aromatase cytochrome P450 messenger ribonucleic acid and enzyme in rat ovarian follicles and corpora lutea: relationship to estradiol biosynthesis.

Gonadotropin-releasing hormone (GnRH) and its analogs have direct extrapituitary effects on the gonads to inhibit steroidogenesis. In the present study, the nondegradable GnRH analog [125I]D-Alaa-des-Gly10-GnRH ethylamide is shown to specifically bind to ovarian, testicular, and adrenal membrane preparations. The ovarian GnRH receptor has a binding affinity similar to that of the anterior pituitary receptor and is specific for GnRH and GnRH analogs. The GnRH-binding capacity of whole ovarian membrane preparations is 150 fm/mg protein, 3- to 4-fold lower than that of anterior pituitary membranes. The number or affinity of receptors prepared from whole ovarian tissue is not influenced by the time of day, stage of the estrous cycle, or hypophysectomy. However, the GnRH receptor concentration is higher in follicular (371 ± 43 fmol/mg) than luteal (162 ± 25 fmol/mg) membranes from cycling rats. Since GnRH injections increase the number of GnRH receptors in the pituitary, the effect of GnRH on ovarian GnRH bind...

Ovarian gonadotropin-releasing hormone (GnRH) receptors: characterization, distribution, and induction by GnRH.

The roles of the pituitary and placenta in pregnant rats and, more specifically, the effects of prolactin and rat placental lactogen on luteal cell function were examined. Hypophysectomy, but not hysterectomy, on day 9 of pregnancy, caused serum progesterone concentrations to decrease 90% and luteal cell LH receptor content to drop 35% by 24 h suggesting pituitary support of luteal cell function at this time. Hypophysectomy and hysterectomy on day 9 effected a similar change in progesterone. The decrease in LH receptor, which was not seen until 48 h, could be prevented by daily administration of prolactin (250 μg/day) or serum of day 12 pregnant rats (2 ml/day). The drop in progesterone production was reduced by the administration of prolactin or PRS alone but was completely prevented by combining prolactin treatment with estradiol (100 μg/day in sesame oil). Hysterectomy on day 12 of pregnancy caused serum progesterone to decrease within 72 h. LH receptor content remained unchanged on day 15 of pregnancy...

Dissociation of two distinct luteotropic effects of prolactin: regulation of luteinizing hormone-receptor content and progesterone secretion during pregnancy.

J. S. Richards

Papers

Ovarian follicular development in the rat: hormone receptor regulation by estradiol follicle stimulating hormone and luteinizing hormone.

Hormonal regulation of ovarian cellular proliferation

Hormonal regulation, tissue distribution, and content of aromatase cytochrome P450 messenger ribonucleic acid and enzyme in rat ovarian follicles and corpora lutea: relationship to estradiol biosynthesis.

Ovarian gonadotropin-releasing hormone (GnRH) receptors: characterization, distribution, and induction by GnRH.

Dissociation of two distinct luteotropic effects of prolactin: regulation of luteinizing hormone-receptor content and progesterone secretion during pregnancy.