121 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1998.In summary, this is the fi... more 121 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1998.In summary, this is the first study to determined that relaxin promotes growth of the vagina during pregnancy in any species. Furthermore, this study also determined that the relaxin-induced growth of the cervix is brought about, in part, by promoting the cellular proliferation of cervical epithelial and stromal cells.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD
Previous results have shown that the pattern of GnRH pulses (amplitude and frequency) can differe... more Previous results have shown that the pattern of GnRH pulses (amplitude and frequency) can differentially regulate expression of gonadotropin subunit cytoplasmic messenger RNA (mRNA) concentrations. The present study examined the effect of GnRH pulses on alpha, LH-beta and FSH-beta transcription rates as determined by nuclear runoff transcription assay. GnRH pulses (saline to controls) were given to castrate, testosterone-replaced male rats, and the rate of subunit gene transcription was measured in isolated pituitary nuclei. The effect of GnRH treatment duration was examined by giving GnRH pulses (25 ng/pulse at 30-min intervals) for 1, 4, or 24 h. The basal transcription rates [expressed as parts per million (ppm)] were 82 +/- 25 for alpha; 39 +/- 19 for LH-beta and 27 +/- 6 ppm for FSH-beta, and transcription rates of all 3 subunits were elevated at 1 h (3-5-fold vs. saline controls). After 4 h of GnRH pulses, alpha and FSH-beta transcription rates were reduced vs. 1 h, but LH-beta mRNA synthesis rate was maintained. At 24 h, the alpha transcription rate was still increased (66%), but LH-beta and FSH-beta transcription rates had fallen to basal levels despite the continuing pulsatile GnRH stimulus. The second experiment investigated the effect of the duration of GnRH pulses (25 ng/pulse, every 30 min for 4 h or 24 h), on cytoplasmic subunit mRNA concentrations to assess if the initial 4-h increase in transcription rate would induce a rise in cytoplasmic mRNAs. After 4 h of GnRH pulses, alpha and LH-beta mRNAs were unchanged, but FSH-beta mRNA had increased by 36% (P less than 0.05) compared to controls. All 3 subunit mRNAs were increased (approximately 2-fold) by 24 h of GnRH pulses. Administering GnRH pulses for 4 h followed by 20 h of saline pulses did not increase alpha mRNA; LH-beta was slightly increased (P less than 0.05), but FSH-beta mRNA concentrations were similar to levels seen after 24 h of continued GnRH pulses. The third experiment examined the effects of a continuous GnRH infusion and different GnRH pulse frequencies on gonadotropin subunit transcription rates. GnRH (25 ng/pulse) was given at intervals of 8, 30, or 120 min for 4 h (saline to controls). The continuous GnRH infusion (200 ng/h) did not increase the transcription rate of any of the three subunit mRNAs. alpha-subunit transcription rate was increased 2.7- or 4-fold by GnRH pulses given every 8 or 30 min, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
The issue of how rapid frequency GnRH pulses selectively stimulate LH transcription is not fully ... more The issue of how rapid frequency GnRH pulses selectively stimulate LH transcription is not fully understood. The rat LHβ promoter contains two GnRH-responsive regions: the proximal region has binding elements for SF1, and the distal site contains a CArG box, which binds SRF. This study determined whether GnRH stimulates pituitary SF1, DAX1 (an endogenous SF1 inhibitor), and SRF transcription in vivo, and whether regulation is frequency dependent. Male rats were pulsed with 25 ng GnRH i.v. every 30 min or every 240 min for 1-24 h, and primary transcripts (PTs) and mRNAs were measured by real time PCR. Fast frequency GnRH pulses (every 30 min) increased SF1 PT (threefold) within 1 h, and then declined after 6 h. SF1 mRNA also increased within 1 h and remained elevated through 24 h. Fast frequency GnRH also stimulated a transient increase in DAX1 PT (twofold after 1 h) and mRNA (1.7-fold after 6 h), while SRF mRNA rose briefly at 1 h. Slow frequency pulses did not affect gene expression of SF1, DAX1, or SRF. These findings support a mechanistic link between SF1 in the frequency regulation of LHβ transcription by pulsatile GnRH.
Polycystic ovary syndrome (PCOS) is the most common form of infertility in women. The causes of P... more Polycystic ovary syndrome (PCOS) is the most common form of infertility in women. The causes of PCOS are not yet understood and both genetics and early-life exposure have been considered as candidates. With regard to the latter, circulating androgens are elevated in mid–late gestation in women with PCOS, potentially exposing offspring to elevated androgens in utero; daughters of women with PCOS are at increased risk for developing this disorder. Consistent with these clinical observations, prenatal androgenization (PNA) of several species recapitulates many phenotypes observed in PCOS. There is increasing evidence that symptoms associated with PCOS, including elevated luteinizing hormone (LH) (and presumably gonadotropin-releasing hormone [GnRH]) pulse frequency emerge during the pubertal transition. We utilized translating ribosome affinity purification coupled with ribonucleic acid (RNA) sequencing to examine GnRH neuron messenger RNAs from prepubertal (3 weeks) and adult female c...
Changes in gonadotropin-releasing hormone (GnRH) release frequency from the brain help drive repr... more Changes in gonadotropin-releasing hormone (GnRH) release frequency from the brain help drive reproductive cycles. In polycystic ovary syndrome (PCOS), persistent high GnRH/luteinizing hormone (LH) frequency disrupts cycles and exacerbates hyperandrogenemia. Adult prenatally-androgenized (PNA) mice exhibit increased GnRH neuron firing rate, elevated ovarian androgens and disrupted cycles, but before puberty GnRH neuron activity is reduced in PNA mice compared to controls. We hypothesized that ovarian feedback mediates the age-dependent change in GnRH neuron firing rate in PNA vs control mice. Extracellular recordings of GFP-identified GnRH neurons were made 5-7d after sham-surgery, ovariectomy (OVX), or, in adults, after OVX plus replacement of sub-male androgen levels with dihydrotestosterone implants (OVX+DHT). In 3wk-old mice, OVX did not affect GnRH neuron firing rate in either group. In adult controls, OVX increased GnRH neuron firing rate, which was further enhanced by DHT. In ...
The brain regulates fertility through gonadotropin-releasing hormone (GnRH) neurons. Estradiol in... more The brain regulates fertility through gonadotropin-releasing hormone (GnRH) neurons. Estradiol induces negative feedback on pulsatile GnRH/luteinizing hormone (LH) release and positive feedback generating preovulatory GnRH/LH surges. Negative and positive feedback are postulated to be mediated by kisspeptin neurons in arcuate and anteroventral periventricular (AVPV) nuclei, respectively. Kisspeptin-specific ERα knockout mice exhibit disrupted LH pulses and surges. This knockout approach is neither location-specific nor temporally-controlled. We utilized CRISPR-Cas9 to disrupt ERα in adulthood. Mice with ERα disruption in AVPV kisspeptin neurons have typical reproductive cycles but blunted LH surges, associated with decreased excitability of these neurons. Mice with ERα knocked down in arcuate kisspeptin neurons showed disrupted cyclicity, associated with increased glutamatergic transmission to these neurons. These observations suggest activational effects of estradiol regulate surge...
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 31, 2018
Estradiol feedback regulates gonadotropin-releasing hormone (GnRH) neurons and subsequent luteini... more Estradiol feedback regulates gonadotropin-releasing hormone (GnRH) neurons and subsequent luteinizing hormone (LH) release. Estradiol acts via estrogen receptor α (ERα)-expressing afferents of GnRH neurons, including kisspeptin neurons in the anteroventral periventricular (AVPV) and arcuate nuclei, providing homeostatic feedback on episodic GnRH/LH release as well as positive feedback to control ovulation. Ionotropic glutamate receptors are important for estradiol feedback, but it is not known where they fit in the circuitry. Estradiol-negative feedback decreased glutamatergic transmission to AVPV and increased it to arcuate kisspeptin neurons; positive feedback had the opposite effect. Deletion of ERα in kisspeptin cells decreased glutamate transmission to AVPV neurons and markedly increased it to arcuate kisspeptin neurons, which also exhibited increased spontaneous firing rate. KERKO mice had increased LH pulse frequency, indicating loss of negative feedback. These observations i...
Reproductive function in mammals is regulated by the pituitary gonadotropins luteinizing hormone ... more Reproductive function in mammals is regulated by the pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH and FSH are secreted by the gonadotrope cell and act on the gonad in a sequential and synergistic manner to initiate sexual maturation and maintain cyclic reproductive function. The synthesis and secretion of LH and FSH are regulated mainly by the pulsatile
American Journal of Physiology - Regulatory, Integrative and Comparative Physiology, 2016
Leptin signals energy sufficiency to the reproductive hypothalamic-pituitary-gonadal (HPG) axis. ... more Leptin signals energy sufficiency to the reproductive hypothalamic-pituitary-gonadal (HPG) axis. Studies using genetic models have demonstrated that hypothalamic neurons are major players mediating these effects. Leptin receptor (LepR) is also expressed in the pituitary gland and in the gonads, but the physiological effects of leptin in these sites are still unclear. Female mice with selective deletion of LepR in a subset of gonadotropes show normal pubertal development but impaired fertility. Conditional deletion approaches, however, often result in redundancy or developmental adaptations, which may compromise the assessment of leptin's action in gonadotropes for pubertal maturation. To circumvent these issues, we adopted a complementary genetic approach and assessed if selective reexpression of LepR only in gonadotropes is sufficient to enable puberty and improve fertility of LepR null female mice. We initially assessed the colocalization of gonadotropin-releasing hormone rece...
Prenatal androgen (PNA) exposure in mice produces a phenotype resembling lean polycystic ovary sy... more Prenatal androgen (PNA) exposure in mice produces a phenotype resembling lean polycystic ovary syndrome (PCOS). We studied effects of voluntary exercise on metabolic and reproductive parameters in PNA vs. vehicle (VEH)-treated mice. Mice (8wks of age) were housed individually and estrous cycles monitored. At 10wk of age, mice were divided into groups (PNA, PNA-run, VEH, VEH-run, n=8-9/group); those in the running groups received wheels allowing voluntary running. Unexpectedly, PNA mice ran less distance than VEH mice; ovariectomy eliminated this difference. In ovary-intact mice there was no difference in glucose tolerance, lower limb muscle fiber types, weight or body composition among groups after 16wks of running, although some mitochondrial proteins were mildly upregulated by exercise in PNA mice. Before running, estrous cycles in PNA mice were disrupted with most days in diestrus. There was no change in cycles during wk1-6 of running (10-15wks of age). In contrast, from wk11-16 ...
The neuropeptide pituitary adenylate cyclase activating polypeptide (ADCYAP 1, or PACAP) has been... more The neuropeptide pituitary adenylate cyclase activating polypeptide (ADCYAP 1, or PACAP) has been demonstrated to enhance gonadotropin-releasing hormone (GnRH)-induced gonadotropin secretion and regulate gonadotropin subunit gene expression in cultures of anterior pituitary cells. In the present study, we used in situ hybridization and real-time polymerase chain reaction to examine the expression of Pacap mRNA within the paraventricular nucleus (PVN) and anterior pituitary throughout the estrous cycle of the rat. Levels of luteinizing hormone in serum and pituitary gonadotropin subunit mRNAs were evaluated and displayed cyclic fluctuations similar to those reported previously. Pacap mRNA expression in the PVN and pituitary varied significantly during the estrous cycle, with the greatest changes occurring on the day of proestrus. Pacap mRNA levels in the PVN declined significantly on the morning of diestrus. During proestrus, PVN Pacap mRNA levels significantly increased 3 h before t...
This study investigated FSHbeta transcriptional responses to the suppression of endogenous follis... more This study investigated FSHbeta transcriptional responses to the suppression of endogenous follistatin (FST) production using FST antisense RNA (FST-AS) expressing adenovirus constructs in female rat pituitary cells in vitro. Adenoviral delivery systems were characterized and optimized using an adenovirus-green fluorescent protein construct, and maximal infection (85-90% of cells) was seen 48 h post adenovirus treatment. A 424 bp fragment, which included the translational start site and exons 1-3 of the rat FST gene, was subcloned in the reverse orientation into an adenovirus vector. Construct efficacy was tested using cultured rat pituitary cells infected with the adenovirus-FST-AS construct. Infection with adenovirus-FST-AS increased FST-AS mRNA expression in a dose-dependent manner, reduced FST protein expression to undetectable levels, and stimulated increases in FSHbeta primary transcript and FSH secretion. Treatment with testosterone alone stimulated FSHbeta primary transcript...
121 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1998.In summary, this is the fi... more 121 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1998.In summary, this is the first study to determined that relaxin promotes growth of the vagina during pregnancy in any species. Furthermore, this study also determined that the relaxin-induced growth of the cervix is brought about, in part, by promoting the cellular proliferation of cervical epithelial and stromal cells.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD
Previous results have shown that the pattern of GnRH pulses (amplitude and frequency) can differe... more Previous results have shown that the pattern of GnRH pulses (amplitude and frequency) can differentially regulate expression of gonadotropin subunit cytoplasmic messenger RNA (mRNA) concentrations. The present study examined the effect of GnRH pulses on alpha, LH-beta and FSH-beta transcription rates as determined by nuclear runoff transcription assay. GnRH pulses (saline to controls) were given to castrate, testosterone-replaced male rats, and the rate of subunit gene transcription was measured in isolated pituitary nuclei. The effect of GnRH treatment duration was examined by giving GnRH pulses (25 ng/pulse at 30-min intervals) for 1, 4, or 24 h. The basal transcription rates [expressed as parts per million (ppm)] were 82 +/- 25 for alpha; 39 +/- 19 for LH-beta and 27 +/- 6 ppm for FSH-beta, and transcription rates of all 3 subunits were elevated at 1 h (3-5-fold vs. saline controls). After 4 h of GnRH pulses, alpha and FSH-beta transcription rates were reduced vs. 1 h, but LH-beta mRNA synthesis rate was maintained. At 24 h, the alpha transcription rate was still increased (66%), but LH-beta and FSH-beta transcription rates had fallen to basal levels despite the continuing pulsatile GnRH stimulus. The second experiment investigated the effect of the duration of GnRH pulses (25 ng/pulse, every 30 min for 4 h or 24 h), on cytoplasmic subunit mRNA concentrations to assess if the initial 4-h increase in transcription rate would induce a rise in cytoplasmic mRNAs. After 4 h of GnRH pulses, alpha and LH-beta mRNAs were unchanged, but FSH-beta mRNA had increased by 36% (P less than 0.05) compared to controls. All 3 subunit mRNAs were increased (approximately 2-fold) by 24 h of GnRH pulses. Administering GnRH pulses for 4 h followed by 20 h of saline pulses did not increase alpha mRNA; LH-beta was slightly increased (P less than 0.05), but FSH-beta mRNA concentrations were similar to levels seen after 24 h of continued GnRH pulses. The third experiment examined the effects of a continuous GnRH infusion and different GnRH pulse frequencies on gonadotropin subunit transcription rates. GnRH (25 ng/pulse) was given at intervals of 8, 30, or 120 min for 4 h (saline to controls). The continuous GnRH infusion (200 ng/h) did not increase the transcription rate of any of the three subunit mRNAs. alpha-subunit transcription rate was increased 2.7- or 4-fold by GnRH pulses given every 8 or 30 min, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
The issue of how rapid frequency GnRH pulses selectively stimulate LH transcription is not fully ... more The issue of how rapid frequency GnRH pulses selectively stimulate LH transcription is not fully understood. The rat LHβ promoter contains two GnRH-responsive regions: the proximal region has binding elements for SF1, and the distal site contains a CArG box, which binds SRF. This study determined whether GnRH stimulates pituitary SF1, DAX1 (an endogenous SF1 inhibitor), and SRF transcription in vivo, and whether regulation is frequency dependent. Male rats were pulsed with 25 ng GnRH i.v. every 30 min or every 240 min for 1-24 h, and primary transcripts (PTs) and mRNAs were measured by real time PCR. Fast frequency GnRH pulses (every 30 min) increased SF1 PT (threefold) within 1 h, and then declined after 6 h. SF1 mRNA also increased within 1 h and remained elevated through 24 h. Fast frequency GnRH also stimulated a transient increase in DAX1 PT (twofold after 1 h) and mRNA (1.7-fold after 6 h), while SRF mRNA rose briefly at 1 h. Slow frequency pulses did not affect gene expression of SF1, DAX1, or SRF. These findings support a mechanistic link between SF1 in the frequency regulation of LHβ transcription by pulsatile GnRH.
Polycystic ovary syndrome (PCOS) is the most common form of infertility in women. The causes of P... more Polycystic ovary syndrome (PCOS) is the most common form of infertility in women. The causes of PCOS are not yet understood and both genetics and early-life exposure have been considered as candidates. With regard to the latter, circulating androgens are elevated in mid–late gestation in women with PCOS, potentially exposing offspring to elevated androgens in utero; daughters of women with PCOS are at increased risk for developing this disorder. Consistent with these clinical observations, prenatal androgenization (PNA) of several species recapitulates many phenotypes observed in PCOS. There is increasing evidence that symptoms associated with PCOS, including elevated luteinizing hormone (LH) (and presumably gonadotropin-releasing hormone [GnRH]) pulse frequency emerge during the pubertal transition. We utilized translating ribosome affinity purification coupled with ribonucleic acid (RNA) sequencing to examine GnRH neuron messenger RNAs from prepubertal (3 weeks) and adult female c...
Changes in gonadotropin-releasing hormone (GnRH) release frequency from the brain help drive repr... more Changes in gonadotropin-releasing hormone (GnRH) release frequency from the brain help drive reproductive cycles. In polycystic ovary syndrome (PCOS), persistent high GnRH/luteinizing hormone (LH) frequency disrupts cycles and exacerbates hyperandrogenemia. Adult prenatally-androgenized (PNA) mice exhibit increased GnRH neuron firing rate, elevated ovarian androgens and disrupted cycles, but before puberty GnRH neuron activity is reduced in PNA mice compared to controls. We hypothesized that ovarian feedback mediates the age-dependent change in GnRH neuron firing rate in PNA vs control mice. Extracellular recordings of GFP-identified GnRH neurons were made 5-7d after sham-surgery, ovariectomy (OVX), or, in adults, after OVX plus replacement of sub-male androgen levels with dihydrotestosterone implants (OVX+DHT). In 3wk-old mice, OVX did not affect GnRH neuron firing rate in either group. In adult controls, OVX increased GnRH neuron firing rate, which was further enhanced by DHT. In ...
The brain regulates fertility through gonadotropin-releasing hormone (GnRH) neurons. Estradiol in... more The brain regulates fertility through gonadotropin-releasing hormone (GnRH) neurons. Estradiol induces negative feedback on pulsatile GnRH/luteinizing hormone (LH) release and positive feedback generating preovulatory GnRH/LH surges. Negative and positive feedback are postulated to be mediated by kisspeptin neurons in arcuate and anteroventral periventricular (AVPV) nuclei, respectively. Kisspeptin-specific ERα knockout mice exhibit disrupted LH pulses and surges. This knockout approach is neither location-specific nor temporally-controlled. We utilized CRISPR-Cas9 to disrupt ERα in adulthood. Mice with ERα disruption in AVPV kisspeptin neurons have typical reproductive cycles but blunted LH surges, associated with decreased excitability of these neurons. Mice with ERα knocked down in arcuate kisspeptin neurons showed disrupted cyclicity, associated with increased glutamatergic transmission to these neurons. These observations suggest activational effects of estradiol regulate surge...
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 31, 2018
Estradiol feedback regulates gonadotropin-releasing hormone (GnRH) neurons and subsequent luteini... more Estradiol feedback regulates gonadotropin-releasing hormone (GnRH) neurons and subsequent luteinizing hormone (LH) release. Estradiol acts via estrogen receptor α (ERα)-expressing afferents of GnRH neurons, including kisspeptin neurons in the anteroventral periventricular (AVPV) and arcuate nuclei, providing homeostatic feedback on episodic GnRH/LH release as well as positive feedback to control ovulation. Ionotropic glutamate receptors are important for estradiol feedback, but it is not known where they fit in the circuitry. Estradiol-negative feedback decreased glutamatergic transmission to AVPV and increased it to arcuate kisspeptin neurons; positive feedback had the opposite effect. Deletion of ERα in kisspeptin cells decreased glutamate transmission to AVPV neurons and markedly increased it to arcuate kisspeptin neurons, which also exhibited increased spontaneous firing rate. KERKO mice had increased LH pulse frequency, indicating loss of negative feedback. These observations i...
Reproductive function in mammals is regulated by the pituitary gonadotropins luteinizing hormone ... more Reproductive function in mammals is regulated by the pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH and FSH are secreted by the gonadotrope cell and act on the gonad in a sequential and synergistic manner to initiate sexual maturation and maintain cyclic reproductive function. The synthesis and secretion of LH and FSH are regulated mainly by the pulsatile
American Journal of Physiology - Regulatory, Integrative and Comparative Physiology, 2016
Leptin signals energy sufficiency to the reproductive hypothalamic-pituitary-gonadal (HPG) axis. ... more Leptin signals energy sufficiency to the reproductive hypothalamic-pituitary-gonadal (HPG) axis. Studies using genetic models have demonstrated that hypothalamic neurons are major players mediating these effects. Leptin receptor (LepR) is also expressed in the pituitary gland and in the gonads, but the physiological effects of leptin in these sites are still unclear. Female mice with selective deletion of LepR in a subset of gonadotropes show normal pubertal development but impaired fertility. Conditional deletion approaches, however, often result in redundancy or developmental adaptations, which may compromise the assessment of leptin's action in gonadotropes for pubertal maturation. To circumvent these issues, we adopted a complementary genetic approach and assessed if selective reexpression of LepR only in gonadotropes is sufficient to enable puberty and improve fertility of LepR null female mice. We initially assessed the colocalization of gonadotropin-releasing hormone rece...
Prenatal androgen (PNA) exposure in mice produces a phenotype resembling lean polycystic ovary sy... more Prenatal androgen (PNA) exposure in mice produces a phenotype resembling lean polycystic ovary syndrome (PCOS). We studied effects of voluntary exercise on metabolic and reproductive parameters in PNA vs. vehicle (VEH)-treated mice. Mice (8wks of age) were housed individually and estrous cycles monitored. At 10wk of age, mice were divided into groups (PNA, PNA-run, VEH, VEH-run, n=8-9/group); those in the running groups received wheels allowing voluntary running. Unexpectedly, PNA mice ran less distance than VEH mice; ovariectomy eliminated this difference. In ovary-intact mice there was no difference in glucose tolerance, lower limb muscle fiber types, weight or body composition among groups after 16wks of running, although some mitochondrial proteins were mildly upregulated by exercise in PNA mice. Before running, estrous cycles in PNA mice were disrupted with most days in diestrus. There was no change in cycles during wk1-6 of running (10-15wks of age). In contrast, from wk11-16 ...
The neuropeptide pituitary adenylate cyclase activating polypeptide (ADCYAP 1, or PACAP) has been... more The neuropeptide pituitary adenylate cyclase activating polypeptide (ADCYAP 1, or PACAP) has been demonstrated to enhance gonadotropin-releasing hormone (GnRH)-induced gonadotropin secretion and regulate gonadotropin subunit gene expression in cultures of anterior pituitary cells. In the present study, we used in situ hybridization and real-time polymerase chain reaction to examine the expression of Pacap mRNA within the paraventricular nucleus (PVN) and anterior pituitary throughout the estrous cycle of the rat. Levels of luteinizing hormone in serum and pituitary gonadotropin subunit mRNAs were evaluated and displayed cyclic fluctuations similar to those reported previously. Pacap mRNA expression in the PVN and pituitary varied significantly during the estrous cycle, with the greatest changes occurring on the day of proestrus. Pacap mRNA levels in the PVN declined significantly on the morning of diestrus. During proestrus, PVN Pacap mRNA levels significantly increased 3 h before t...
This study investigated FSHbeta transcriptional responses to the suppression of endogenous follis... more This study investigated FSHbeta transcriptional responses to the suppression of endogenous follistatin (FST) production using FST antisense RNA (FST-AS) expressing adenovirus constructs in female rat pituitary cells in vitro. Adenoviral delivery systems were characterized and optimized using an adenovirus-green fluorescent protein construct, and maximal infection (85-90% of cells) was seen 48 h post adenovirus treatment. A 424 bp fragment, which included the translational start site and exons 1-3 of the rat FST gene, was subcloned in the reverse orientation into an adenovirus vector. Construct efficacy was tested using cultured rat pituitary cells infected with the adenovirus-FST-AS construct. Infection with adenovirus-FST-AS increased FST-AS mRNA expression in a dose-dependent manner, reduced FST protein expression to undetectable levels, and stimulated increases in FSHbeta primary transcript and FSH secretion. Treatment with testosterone alone stimulated FSHbeta primary transcript...
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