PUMA regulates germ cell loss and primordial follicle endowment in mice.

The number of primordial follicles initially established within the ovary is influenced by the extent of germ cell death during foetal ovarian development, but the mechanisms that mediate this death have not been fully uncovered. In this study, we identified BBC3 (PUMA) (p53 upregulated modulator of apoptosis, also known as BCL2-binding component 3), a pro-apoptotic BH3-only protein belonging to the BCL2 family, as a critical determinant of the number of germ cells during ovarian development. Targeted disruption of the Bbc3 gene revealed a significant increase in the number of germ cells as early as embryonic day 13.5. The number of germ cells remained elevated in Bbc3(-/-) female mice compared with WT female mice throughout the remainder of embryonic and early postnatal life, resulting in a 1.9-fold increase in the number of primordial follicles in the ovary on postnatal day 10. The increase in the number of germ cells observed in the ovaries of Bbc3(-/-) mice could not be attributed to the altered proliferative activity of germ cells within the ovaries. Furthermore, BBC3 was found to be not required for the massive germ cell loss that occurs during germ cell nest breakdown. Our data indicate that BBC3 is a critical regulator of germ cell death that acts during the migratory phase of oogenesis or very soon after the arrival of germ cells in the gonad and that BBC3-mediated cell death limits the number of primordial follicles established in the initial ovarian reserve.

Molecular correlates of platinum response in human high-grade serous ovarian cancer patient-derived xenografts.

INTRODUCTION: Improvement in the ability to target underlying drivers and vulnerabilities of high-grade serous ovarian cancer (HG-SOC) requires the development of molecularly annotated pre-clinical models reflective of clinical responses. METHODS: We generated patient-derived xenografts (PDXs) from consecutive, chemotherapy-naïve, human HG-SOC by transplanting fresh human HG-SOC fragments into subcutaneous and intra-ovarian bursal sites of NOD/SCID IL2Rγ(null) recipient mice, completed molecular annotation and assessed platinum sensitivity. RESULTS: The success rate of xenografting was 83%. Of ten HG-SOC PDXs, all contained mutations in TP53, two were mutated for BRCA1, three for BRCA2, and in two, BRCA1 was methylated. In vivo cisplatin response, determined as platinum sensitive (progression-free interval ≥ 100 d, n = 4), resistant (progression-free interval <100 d, n = 3) or refractory (n = 3), was largely consistent with patient outcome. Three of four platinum sensitive HG-SOC PDXs contained DNA repair gene mutations, and the fourth was methylated for BRCA1. In contrast, all three platinum refractory PDXs overexpressed dominant oncogenes (CCNE1, LIN28B and/or BCL2). CONCLUSIONS: Because PDX platinum response reflected clinical outcome, these annotated PDXs will provide a unique model system for preclinical testing of novel therapies for HG-SOC.

The dynamics of the primordial follicle reserve.

The female germline comprises a reserve population of primordial (non-growing) follicles containing diplotene oocytes arrested in the first meiotic prophase. By convention, the reserve is established when all individual oocytes are enclosed by granulosa cells. This commonly occurs prior to or around birth, according to species. Histologically, the 'reserve' is the number of primordial follicles in the ovary at any given age and is ultimately depleted by degeneration and progression through folliculogenesis until exhausted. How and when the reserve reaches its peak number of follicles is determined by ovarian morphogenesis and germ cell dynamics involving i) oogonial proliferation and entry into meiosis producing an oversupply of oocytes and ii) large-scale germ cell death resulting in markedly reduced numbers surviving as the primordial follicle reserve. Our understanding of the processes maintaining the reserve comes primarily from genetically engineered mouse models, experimental activation or destruction of oocytes, and quantitative histological analysis. As the source of ovulated oocytes in postnatal life, the primordial follicle reserve requires regulation of i) its survival or maintenance, ii) suppression of development (dormancy), and iii) activation for growth and entry into folliculogenesis. The mechanisms influencing these alternate and complex inter-related phenomena remain to be fully elucidated. Drawing upon direct and indirect evidence, we discuss the controversial concept of postnatal oogenesis. This posits a rare population of oogonial stem cells that contribute new oocytes to partially compensate for the age-related decline in the primordial follicle reserve.

DNA damage-induced primordial follicle oocyte apoptosis and loss of fertility require TAp63-mediated induction of Puma and Noxa.

Trp63, a transcription factor related to the tumor suppressor p53, is activated by diverse stimuli and can initiate a range of cellular responses. TAp63 is the predominant Trp53 family member in primordial follicle oocyte nuclei and is essential for their apoptosis triggered by DNA damage in vivo. After γ-irradiation, induction of the proapoptotic BH3-only members Puma and Noxa was observed in primordial follicle oocytes from WT and Trp53(-/-) mice but not in those from TAp63-deficient mice. Primordial follicle oocytes from mice lacking Puma or both Puma and Noxa were protected from γ-irradiation-induced apoptosis and, remarkably, could produce healthy offspring. Hence, PUMA and NOXA are critical for DNA damage-induced, TAp63-mediated primordial follicle oocyte apoptosis. Thus, blockade of PUMA may protect fertility during cancer therapy and prevent premature menopause, improving women's health.

Cysteine-rich secretory proteins are not exclusively expressed in the male reproductive tract.

The Cysteine-RIch Secretory Proteins (CRISPs) are abundantly produced in the male reproductive tract of mammals and within the venom of reptiles and have been shown to regulate ion channel activity. CRISPs, along with the Antigen-5 proteins and the Pathogenesis related-1 (Pr-1) proteins, form the CAP superfamily of proteins. Analyses of EST expression databases are increasingly suggesting that mammalian CRISPs are expressed more widely than in the reproductive tract. We, therefore, conducted a reverse transcription PCR expression profile and immunohistochemical analyses of 16 mouse tissues to define the sites of production of each of the four murine CRISPs. These data showed that each of the CRISPs have distinct and sometimes overlapping expression profiles, typically associated with the male and female reproductive tract, the secretory epithelia of exocrine glands, and immune tissues including the spleen and thymus. These investigations raise the potential for a role for CRISPs in general mammalian physiology.

Histologic and ultrastructural evaluation of sutures used for surgical fixation of the SMAS.

In extensive SMAS face-lift surgery, retaining ligaments are released, and the SMAS is resutured to the deep fascia to maintain the advanced position. The suture used to reattach the SMAS should replicate the quality of support provided by the original ligaments. Nonabsorbable sutures (monofilament and braided) retrieved intraoperatively from 22 patients undergoing secondary face-lift procedures were examined by light microscopy and transmission electronmicroscopy. A distinctive enclosure of dense collagen and elastin formed around both types of suture. Based on the presence of inflammatory cells, fibroblasts, collagen, and elastin, the tissue reaction to monofilament suture was less than with the braided suture. The collagen and elastin were thicker around the braided suture, and, additionally the collagen matrix infiltrated between the individual filaments. Ultrastructural analysis of the braided suture showed significant collagen binding around each individual filament. The greater quantity of connective tissue around the thread which continued into the interstices of the braided suture has the characteristics of a ligament. This suggests a stronger and more lasting tissue fixation.

The hypogonadal (hpg) mouse as a model to investigate the estrogenic regulation of spermatogenesis.

The hypogonadal (hpg) mouse is an excellent animal model in which to investigate the mechanism of action of estrogens on spermatogenesis because it has arrested reproductive development without the need for surgical, endocrine, pharmacological or immunological intervention. Hpg mice are hypogonadotrophic and fail to show normal postnatal testicular development due to the congenital inability to synthesize gonadotropin-releasing hormone in the hypothalamus. The hpg testis remains responsive to gonadotropins and androgens in that fertility can be induced by treatment with these hormones. Surprisingly, chronic treatment with low concentrations of estradiol alone induces qualitatively normal spermatogenesis. The induction of testicular development by estradiol in hpg mice is accompanied by a paradoxical increase in FSH production. The actions of estradiol in hpg mice appear to be via genomic estrogen receptors, as concurrent treatment with estrogen-receptor antagonist ICI182,780 completely blocks these pituitary and testis responses. Concurrent treatment with the androgen receptor antagonist bicalutamide does not affect the estradiol-induced increase in pituitary FSH content, but markedly attenuates the estradiol-induced increase in testicular weight. Western blot analyses and immunohistochemistry provide evidence for estrogen-receptor alpha and beta expression in both pituitary gland and testis of the hpg mouse. Estradiol may therefore exert direct actions within the testes and/or indirect neuroendocrine actions via the release of FSH or other hormones from the pituitary gland, but its actions are dependent upon the availability of low levels of androgen within the testis.

Sonography of plantar plates in cadavers: correlation with MRI and histology.

OBJECTIVE: The purpose of our study was to describe the sonographic appearance of the lesser metatarsal plantar plates in cadavers and to correlate these findings with MRI and histology. MATERIALS AND METHODS: Six soft-embalmed cadaveric feet (74-92 years old; two male, one female) were imaged with sonography and MRI. Tear dimensions of the plantar plate were recorded in the long and short axes. Orthopedic surgeons directly inspected the plantar plates before removing samples for histologic correlation. One young fresh cadaver was imaged with sonography before histologic assessment. RESULTS: The normal plantar plate appearance on sonography was a slightly echoic, homogeneous, curved structure. At direct inspection, a tear was present in 23 (96%) of 24 of the lesser plantar plates in the soft-embalmed feet. This direct inspection correlated with sonography detecting 23 tears correctly and MRI, 22 tears. Both sonography and MRI falsely reported one tear, but MRI also failed to detect one tear. Histologically, the abnormal plantar plate showed loss of the normal dense regular tissue and replacement with vessels, hydropic tissue, and a mixture of loose connective tissue and dense irregular connective tissue. CONCLUSION: Sonography, being noninvasive, shows promise as an imaging tool of the plantar plate. With ongoing research in this area we hope to determine the reliability and significance of such a technique in the evaluation of the plantar plate.

Estrogenic induction of spermatogenesis in the hypogonadal (hpg) mouse: role of androgens.

Testicular development is arrested in the hypogonadal (hpg) mouse due to a congenital deficiency of hypothalamic gonadotropin-releasing hormone synthesis. Previous studies have demonstrated that chronic treatment of these mice with estradiol induces testicular maturation and qualitatively normal spermatogenesis, but it is not known whether these are direct effects via estrogen receptors expressed in the testis, or indirect actions via the pituitary gland. The aim of the current studies was to determine whether the actions of estradiol require the presence of androgens. Sensitive assays revealed that chronic estradiol treatment produced time-dependent increases in pituitary FSH production but no increases in pituitary LH or testicular testosterone content could be detected. As a functional test of androgen dependence, hpg mice were treated for 70 days with estradiol plus Casodex (bicalutamide), an androgen receptor antagonist. Casodex treatment markedly attenuated both the estradiol-induced increase in testicular weight and the proliferation of the seminiferous epithelium, as revealed by morphometric analysis. However, it did not affect the estradiol-induced increase in pituitary FSH content, nor did it affect estradiol-induced increases in the weight of the seminal vesicles and epididymides. We conclude that increased FSH production is not sufficient to explain the increase in testicular development induced by estradiol in hpg mice; there is a requirement for functional androgen receptors for induction of testicular growth.

Neonatal androgenization of hypogonadal (hpg) male mice does not abolish estradiol-induced FSH production and spermatogenesis.

BACKGROUND: Testicular development is arrested in the hypogonadal (hpg) mouse due to a congenital deficiency in hypothalamic gonadotropin-releasing hormone (GnRH) synthesis. Chronic treatment of male hpg mice with estradiol induces FSH synthesis and secretion, and causes testicular maturation and qualitatively normal spermatogenesis. As estradiol negative feedback normally inhibits FSH production in the male, this study tested whether this paradoxical response to estradiol in the male hpg mouse might be due to inadequate masculinisation or incomplete defeminization in the neonatal period. Previous studies have demonstrated that treatment of hpg mice with testosterone propionate in the immediate neonatal period is necessary to allow full reproductive behaviors to be expressed following suitable endocrine stimulation at adult ages. METHODS: Hpg mice were treated with 100 mug testosterone propionate or vehicle on postnatal day 2. At 35 days of age, subgroups of these mice were treated with silastic implants containing estradiol or cholesterol. Reproductive behavior was scored in tests with steroid-primed female mice, then testicular development was assessed histologically, and measures of pituitary FSH content made at 85 days of age. RESULTS: The neonatal testosterone propionate treatment successfully defeminized female litter mates, as revealed by impaired vaginal opening and deficiencies in lordosis behavior, and it allowed appropriate male reproductive behavior to be expressed in a proportion of the hpg males when tested at an adult age. However, neonatal androgen supplementation did not block or even reduce the subsequent actions of estradiol in increasing pituitary FSH content, nor did it affect the ability of estradiol to induce qualitatively normal spermatogenesis. CONCLUSION: The ability of the hpg male to show a "female" neuroendocrine response to estradiol is not a result of inadequate androgenization during neonatal development, and thus the actions of estradiol revealed in this rodent model are not an artefact of incomplete sexual differentiation, but reflect a physiological role of estradiol occurring during a specific early temporal window of male reproductive development.