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K J Barr

Publications and source records attributed to K J Barr.

13 recordsLinked to original sources
PubMed1996

Multiple members of the connexin gene family participate in preimplantation development of the mouse.

The connexin gene family, of which there are at least 12 members in rodents, encodes the protein subunits intercellular membrane channels (gap junction channels). Because of the diverse structural and biophysical properties exhibited by the different connexins, it has been proposed that each may play a unique role in development or homeostasis. We have begun to test this hypothesis in the preimplantation mouse embryo in which de novo gap junction assembly is a developmentally regulated event. As a first step, we have used reverse transcription-polymerase chain reaction (RT-PCR) to determine the connexin mRNA phenotype of mouse blastocysts, and have identified transcripts of connexins 30.3, 31, 31.1, 40, 43, and 45. Quantitative measurements indicated that all six of these connexin genes are transcribed after fertilization. They can be divided into two groups with respect to the timing of mRNA accumulation: Cx31, Cx43, and Cx45 mRNAs accumulate continuously from the two- or four-cell stage, whereas Cx30.3, Cx31.1, and Cx40 mRNAs accumulate beginning in the eight-cell stage. All six mRNAs were found to co-sediment with polyribosomes from their time of first appearance, indicating that all six are translated. The expression of Cx31.1 and Cx40 was examined by confocal immunofluorescence microscopy; whereas both could be detected in compacting embryos, only Cx31.1 could be seen in punctate membrane foci indicative of gap junctions. Taken together with other results (published or submitted), our findings indicate that at least four connexins (Cx31, 31.1, 43 and 45) contribute to gap junctions in preimplantation development. The expression of multiple connexin genes during this early period of embryogenesis (when there are only two distinct cell types) raises questions about the functional significance of connexin diversity in this context.

Animals↗
PubMed1994

Regulation of Na+,K(+)-ATPase alpha subunit gene expression during mouse preimplantation development.

Several lines of evidence support the hypothesis that a sodium flux, driven by Na+,K(+)-ATPase in the basolateral plasma membranes of mural trophectoderm, drives fluid transport during blastocoel formation in eutherians. In light of the importance of this enzyme for preimplantation development, attention has been focused on the regulation of expression of its alpha and beta subunits. Here we report on the spatial distribution and translation of the alpha subunit mRNA. Although this mRNA accumulates from the 2-cell stage onward the alpha subunit itself could not be detected by immunofluorescence prior to the late morula stage, after which it becomes concentrated in the mural trophectoderm. In the present study we have used a wholemount, fluorescent in situ hybridization technique that takes advantage of the optical sectioning capability of the confocal microscope to show that alpha subunit mRNA, in contrast to the alpha subunit itself, accumulates in all cells of the early blastocyst. This finding demonstrates that the spatial distribution of the alpha subunit is regulated post-transcriptionally. We have also examined the translational regulation of alpha subunit mRNA by preparing polyribosomal and subribosomal ribonucleoprotein fractions for mRNA assay by reverse transcription-polymerase chain reaction. We found that alpha subunit mRNA is in polyribosomes continuously from at least the 4-cell stage. Thus, the abrupt appearance of the alpha subunit in the late morula stage as revealed by immunofluorescence must be determined by post-translational events. In the Discussion, we consider the hypothesis that synthesis of the beta subunit of the enzyme is the rate limiting step in functional expression of the alpha subunit.

Animals↗
PubMed1991

A radioimmunoassay for the regulatory allylic steroid, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP).

The allylic steroid, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP), found in gonadal and brain tissues by radiotracer and chemical methods, had been shown to play a role in gametogenesis, gonadotropin secretion and brain excitability. Since no simple assay was available, a radioimmunoassay for 3 alpha HP was developed using [3H]3 alpha HP and an antiserum raised against 3 alpha HP-20-CMO conjugated to bovine serum albumin. The specificity of the assay for the 3 alpha allylic configuration of 3 alpha HP was confirmed by examining 32 other steroids; cross-reaction with steroids containing different configurations (including metabolites of 3 alpha HP such as progesterone) was less than 0.9%. A Scatchard plot indicated a Ka of 1.56 X 10(9) M-1. Inter- and intra-assay coefficients of variation were 13.1 and 4.5%, respectively. The sensitivity of the assay was 6 pg and the 50% intercept of the standard curve was approx. 123 pg. The measurement by RIA of 3 alpha HP from standard solutions and HPLC purified tissue extracts was confirmed qualitatively and quantitatively by GC/MS methods. The RIA method was employed to determine 3 alpha HP levels in cultured Sertoli cells and in serum of intact and ovariectomized adult rats. Although for most uses, chromatography would not be necessary, two possible methods are presented to enable the separation of 3 alpha HP from other interfering steroids prior to RIA.

20-alpha-Dihydroprogesterone↗
PubMed1989

Sustained azoospermia in squirrel monkey, Saimiri sciureus, resulting from a single intratesticular glycerol injection.

Previous studies in rats had shown that a single intratesticular injection of glycerol resulted in long-term suppression of spermatogenesis without marked alterations in hormone levels. Studies were undertaken to determine the effect of similar treatment in squirrel monkeys (Saimiri sciureus). Ten monkeys received an intratesticular injection of saline (controls) and ten of glycerol solution (treated). Semen and blood samples were obtained on a weekly or bi-weekly basis one month prior to, during the 8 months following and at 22 months after the injection. Sperm numbers in the semen samples of controls remained at 160-435 x 10(6) per ml throughout the experiment. Sperm numbers in treated animals declined to near zero within two months and remained at zero. Serum testosterone and progesterone levels were not significantly different between control and treated animals. Serum LH and FSH levels were not significantly different between control and treated animals except during months 6-8 after the injection, when levels in the treated were higher. At termination (22 months), the weights and sperm contents of epididymides of the glycerol-treated animals were highly significantly reduced. Steroidogenesis (based on amounts and kinds of steroids formed from 14C-progesterone) by testicular tissue was not altered by the glycerol treatment when measured on a per testis basis. This is the first evidence that a single intratesticular injection of glycerol results in long-term suppression of spermatogenesis in primates, without altering testicular steroidogenesis and serum hormone levels.

Androgens↗
PubMed1988

Effect of prenatal and neonatal exposure to lead on the affinity and number of estradiol receptors in the uterus.

Female Sprague-Dawley rats were treated with lead chloride (20 ppm or 200 ppm Pb) or sodium chloride (controls) in their drinking water. Three treatment regimens were employed: (I) rats were treated prior to mating and uteri were removed from 21-d-old offspring, (II) treatments were begun when females were in d 7 of pregnancy and continued on the dams until the pups were 21 d old, and half of these offspring were continued on the Pb treatments and half on saline, with uteri removed during diestrus when female offspring were approximately 150 d old; (III) female rats were treated from d 21 to d 35 and then uteri were removed. Estradiol-receptor binding and affinity were determined on the uterine tissues. Treatment with lead prior to mating (group I) resulted in a significant increase in estradiol-receptor affinity (Ka) in 21-d-old offspring without a change in estradiol receptor number (N). Treatment from d 7 of pregnancy until weaning of the pups resulted in approximately 35% decrease (p less than 0.05) in estradiol receptors per milligram uterine protein when these offspring reached 150 d of age (group II). Similarly, treatment with Pb from d 21 until d 35 or until d 150 resulted in a significant decrease in uterine estradiol receptor number at 35 and 150 d, respectively, while the Ka was significantly (p less than 0.01) increased by the exposure to Pb. The results demonstrate that prenatal and/or postnatal exposure to Pb alters the number and affinity of estradiol receptors in the prepubertal and adult rat uterus.

Animals↗
PubMed1988

Effect of prenatal and neonatal exposure to lead on gonadotropin receptors and steroidogenesis in rat ovaries.

Sprague-Dawley rats were treated with lead chloride (20 or 200 ppm) or sodium chloride (controls) in their drinking water, either prior to pregnancy or during pregnancy and lactation, and female offspring were examined at weaning (21 d) or at 150 d. Other female rats were treated from d 21 to 35. Tissue (blood, kidney, bone) lead levels, body, ovary, and uterus weights, ovarian steroidogenesis, and gonadotropin (luteinizing hormone and follicle-stimulating hormone) levels, and gonadotropin-receptor binding were determined. Prenatal and/or postnatal exposure to lead at these levels (20 and 200 ppm) did not affect tissue weights but did cause a significant decrease in gonadotropin-receptor binding in the prepubertal, pubertal and adult females. Conversion of progesterone to androstenedione and dihydrotestosterone was significantly decreased in 21-d-old rats; in 150-d-old females, the prenatal and/or postnatal exposure to lead resulted in significantly increased conversion to the 5-alpha-reduced steroids, normally high during puberty. The results demonstrate that lead exposure prior to mating may affect gonadotropin-receptor binding in the offspring and that lead exposure (in utero, via mother's milk, or post weaning) may significantly alter steroid production and gonadotropin binding in ovaries of the prepubertal, pubertal, and adult female.

Animals↗
PubMed1984

Suppression of spermatogenesis without inhibition of steroidogenesis by a 1,2,3-trihydroxypropane solution.

This study presents the first evidence that the non-hormonal, biological substance, 1,2,3- trihydroxypropane (THP) acts as a selective and potent antispermatogenic agent without any apparent toxic or endocrine side effects. The studies were done on 119 rats which were injected intratesticularly (50-200 microliter) with either sterile filtered distilled water (Control) or water/THP (3:7; Treated) and histological, biochemical and fertility determinations were made up to 11 weeks after the injections. The results show that within one week of a single injection with THP, the weights of testes and epididymides are significantly less than those of the Controls and the reduced weights persist for at least 11 weeks. The seminiferous tubules are largely depleted of spermatogenetic cells by 2 weeks and they remain devoid of dividing germ cells for the 11 week period. The Leydig cells have a normal appearance and histochemically show the same 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) and 3 beta-HSD activities as Controls. In vitro studies of the testes showed that the activities of the steroid enzymes 20 alpha-HSD, 17 beta-HSD, 17 alpha-hydroxylase and C17-20-lyase and the production of testosterone and androstenedione were not altered by the THP treatment. Similarly, serum levels of testosterone, LH, and FSH, measured by RIA, and weights of the prostate and seminal vesicles were the same as in Controls. Treated males showed the same degree of sexual behaviour and mating frequency as the Controls, but after the 3rd mating were 100% infertile for the duration of the experiments. The total number of sperm in epididymides of Treated rats was reduced by 99.99% after the 3rd mating.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

The control of male fertility by 1,2,3-trihydroxypropane (THP;glycerol): rapid arrest of spermatogenesis without altering libido, accessory organs, gonadal steroidogenesis, and serum testosterone, LH and FSH.

This study examines the effects of intratesticular injection of aqueous 1,2,3-trihydroxypropane (THP; glycerol) solution on male reproductive biology. In a series of experiments, Sprague-Dawley rats of various ages (48-101 days) were injected with 50-200 microliters per testis and various parameters were studied for up to 21 weeks later. While an injection of THP resulted in testicular weight reduction of 45-60% within 2 weeks, the weights of prostate and seminal vesicles were not affected for the duration of the experiments. The number of sperm per epididymis in the THP-treated rats declined rapidly and was reduced by 99.99% (of controls) after the 3rd mating. THP-treated males mated with virgin females at the same frequency as control rats but all were infertile after the 3rd mating and remained infertile for the duration of the tests (21 weeks after treatment). In vitro studies showed that metabolism of 14C-progesterone by testicular homogenates was not altered quantitatively or qualitatively by THP treatment. Serum levels of androgens, LH and FSH of THP-treated rats did not differ significantly from the controls. Histologically and histochemically, the Leydig cells appeared to be normal, but the seminiferous tubules of THP-treated testes were devoid of spermatogenic cells within 2 weeks of a single treatment. It is concluded that direct injection of THP acts as a potent inhibitor of spermatogenesis resulting in long-term infertility without affecting steroidogenesis, libido, secondary sex characteristics, mating behaviour or serum hormone levels.

Animals↗
PubMedDate not supplied

Lead administration during pregnancy and lactation affects steroidogenesis and hormone receptors in testes of offspring.

Sprague-Dawley rats were injected sc with lead acetate in suspension, or with sodium acetate (controls) on the d 9 of pregnancy and every 3-4 d thereafter until the pups were 13 or 21 d of age. At termination, testicular homogenates or isolated Sertoli cells were used to study steroidogenesis and gonadotropin binding. Lead had no significant effect on the mother's water and food consumption, on the pup's body or testis weights, on the number of pups and the time of birth, and on the seminiferous tubule diameter. Homogenates of testes of the lead-treated group converted significantly less (p less than 0.01) labeled progesterone (14C or 3H) to 5 alpha-pregnane-3,20-dione, 3 alpha-hydroxy-5 alpha-pregnan-20-one, 17 beta-hydroxy-5 alpha-androstan-3-one (DHT), 3(alpha, beta)-hydroxy-5 alpha-androstan-17-one, testosterone/17 alpha-hydroxyprogesterone, and androstenedione. Sertoli cells from lead-treated animals converted significantly less (p less than 0.01) progesterone to 5 alpha-pregnane-3 alpha, 20 alpha-diol, 3 alpha-hydroxy-5 alpha-pregnan-20-one, DHT, and 20 alpha-hydroxy-4-pregnen-3-one. These data and direct spectrophotometric assays indicated that 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSO), 3 beta-HSO, 20 alpha-HSO, 5 alpha-reductase, and C17-20-lyase had been affected. The receptor studies showed that the binding of [125I]rFSH to testicular receptors was significantly reduced from 35,600 (control) to 25,980 cpm/mg protein (lead). This is the first evidence that lead exposure (in utero and via mother's milk) significantly reduces steroid production and hormone binding in the testis at the onset of puberty.

Animals↗