an-improved-time-labor-efficient-protocol-for-mouse-primary

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...

Seattle Children’s Hospital

Androgen and its receptor (AR) play a critical role in reproductive function under both physiological and pathophysiological conditions. Female AR global knockout mice are subfertile due to both neuroendocrine and ovarian defects. Female offspring from prenatally androgenized heterozygous AR pregnant mice showed rescued estrous cyclicity and fertility. Ar is expressed in granulosa cells, theca interstitial cells, and oocytes in the ovary. We created mice with theca-specific deletion of Ar (ThARKO) by crossing Cyp17-iCre mice that express Cre recombinase under cytochrome P450 17A1 (Cyp17) promoter with Arfl/fl mice. ThARKO mice exhibited no significant differences in pubertal onset or fertility compared with control littermates, and neither estrogen or testosterone levels were different between these groups. Therefore, Ar expression in theca cells likely does not influence fertility nor androgen levels in female mice. We then tested the role of AR in theca cells under hyperandrogenemic condition. After treatment with a pathophysiological level of dihydrotestosterone (DHT), control mice (control-DHT) showed acyclicity and infertility. However, estrous cycles and fertility were altered to a significantly less degree in ThARKO-DHT mice than in control-DHT mice. Messenger RNA (mRNA) levels of Lhcgr (luteinizing hormone receptor) and Timp1 (tissue inhibitor of metalloproteinase 1, and inhibitor of matrix metalloproteinase) were significantly lower in control-DHT ovary compared with control-no DHT ovaries, whereas mRNA levels of Fshr (follicle-stimulating hormone receptor) were significantly higher. Timp1 gene expression was comparable in the ThARKO-DHT and the control-no DHT ovary. We speculate that the preserved level of Timp1 in ThARKO-DHT mice contributes to retained reproductive function.

Androgen Receptor in the Ovary Theca Cells Plays a Critical Role in Androgen-Induced Reproductive Dysfunction.

Female offspring of many species exposed to high doses of androgens in utero experience endocrine dysfunction during adulthood. The phenotype of offspring from females with prepregnancy hyperandrogenemia and impaired ovulation, however, has not been examined. We developed a mouse model of hyperandrogenemia by implanting a low-dose dihydrotestosterone (DHT) pellet 15 days before conception. Female offspring born to dams with hyperandrogenemia (DHT daughters) had delayed puberty (P < 0.05) with first estrus on postnatal day (PND) 41 compared with daughters from dams with physiological levels of DHT (non-DHT daughters, PND37.5). Serum follicle-stimulating hormone (FSH) levels in the DHT daughters were fourfold higher (P < 0.05) on PND21, and anti-Müllerian hormone levels were higher (P < 0.05) on PND26 than in non-DHT daughters (controls). DHT daughters showed an extended time in metestrus/diestrus and a shorter time in the proestrus/estrus phase compared with non-DHT daughters (P < 0.05). To examine ovarian response to gonadotropins, superovulation was induced and in vitro fertilization (IVF) was performed. Fewer numbers of oocytes were retrieved from the DHT daughters compared with non-DHT daughters (P < 0.05). At IVF, there was no difference in rates of fertilization or cleavage of oocytes from either group. There were fewer (P < 0.01) primordial follicles (6.5 ± 0.8 vs 14.5 ± 2.1 per ovary) in the ovaries of DHT daughters compared with non-DHT daughters. Daughters from hyperandrogenemic females exhibited elevated prepubertal FSH levels, diminished ovarian response to superovulation, impaired estrous cyclicity, delayed onset of puberty, and reduced ovarian reserve, suggesting that fetal androgen exposure had lasting effects on female reproductive function.

Female Offspring From Chronic Hyperandrogenemic Dams Exhibit Delayed Puberty and Impaired Ovarian Reserve.

Care of transgender and gender diverse youth is complex and requires a multidisciplinary approach. Many transgender patients and providers feel the limited availability of affirming, knowledgeable professionals is a barrier to obtaining care. Such care can be provided through a clinic with providers from different disciplines who are trained in the unique care of transgender youth. In this paper, we discuss the care guidelines for transgender youth and the unresolved challenges that need to be addressed during the development of a transgender clinic. We describe our experience at Seattle Children's Hospital in the development of a multidisciplinary Gender Clinic which incorporates the expertise of social work, mental health professionals, pediatric endocrinology, adolescent medicine, and bioethics. Other institutions may build from our experience, with the ultimate goal of further decreasing health disparities for young transgender patients.

Review of Current Care Models for Transgender Youth and Application to the Development of a Multidisciplinary Clinic - The Seattle Children's Hospital Experience.

 To assess youth and parent/caregiver satisfaction with care at a pediatric multidisciplinary gender clinic.  Transgender/gender nonconforming youth (=33) and their parent/caregiver (=29) completed self-report questionnaires and individual interviews (=20) about experiences and satisfaction with care.  Quantitatively, participants reported being extremely satisfied with care experiences (parents 97%; youth 94%). Qualitatively, main themes included (1) affirmation due to use of preferred name/pronouns, (2) access barriers due to scheduling and readiness assessments, and (3) positive interactions with Care Navigator.  Youth and parents/caregivers are highly satisfied with multidisciplinary, coordinated health care for transgender/gender nonconforming youth; however, some challenges remain.

Youth and Parent Experiences in a Multidisciplinary Gender Clinic.

To discuss treatments used to enhance growth in pediatric patients with short stature.

Sara Divall

Department of Pediatrics

Seattle Children’s Hospital

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Sara Divall

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pediatrics

Seattle Children’s Hospital

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Department of Pediatrics