Production of monoclonal antibodies against recombinant human zona pellucida glycoproteins: utility in immunolocalization of respective zona proteins in ovarian follicles.

The zona pellucida (ZP) glycoproteins play an important role in oocyte development and gamete biology. To analyze their expression in follicles during various developmental stages, murine monoclonal antibodies (MAbs) were generated against the baculovirus-expressed recombinant human ZP2, ZP3 and ZP4. A panel of MAbs specific for the respective zona protein in ELISA and Western blot, and devoid of cross-reaction with other zona proteins was selected. Immunohistochemistry has shown that ZP2 MAb, MA-1620, did not react with oocytes in resting primordial follicles but showed reactivity with degenerating oocytes in primordial follicles undergoing atresia, and with oocytes in growing and antral follicles. Three MAbs against ZP3 did not react with oocytes in primordial follicles, but reacted only with oocytes in growing and antral follicles. Out of four MAbs against ZP4, three MAbs reacted with oocytes in primordial, growing and antral follicles. No reactivity of these MAbs with other ovarian cell types and other tissues studied (endometrium, uterine cervix, fallopian tubes and kidney) was detected except for a strong reactivity of ZP2 MA-1620 with epithelial cells of the uterine ectocervix or endometrium in some samples investigated. Altogether, these studies document generation of MAbs exhibiting high specificity for human zona proteins, which will be useful reagents to study their immunobiology.

Study origin of germ cells and formation of new primary follicles in adult human and rat ovaries.

The central thesis regarding the human ovaries is that, although primordial germ cells in embryonal ovaries are of extraovarian origin, those generated during the fetal period and in postnatal life are derived from the ovarian surface epithelium (OSE) bipotent cells. With the assistance of immune system-related cells, secondary germ cells and primitive granulosa cells originate from OSE stem cells in the fetal and adult human gonads. Fetal primary follicles are formed during the second trimester of intrauterine life, prior to the end of immune adaptation, possibly to be recognized as self-structures and renewed later. With the onset of menarche, a periodical oocyte and follicular renewal emerges to replace aging primary follicles and ensure that fresh eggs for healthy babies are always available during the prime reproductive period. The periodical follicular renewal ceases between 35 and 40 yr of age, and the remaining primary follicles are utilized during the premenopausal period until exhausted. However, the persisting oocytes accumulate genetic alterations and may become unsuitable for ovulation and fertilization. The human OSE stem cells preserve the character of embryonic stem cells, and they may produce distinct cell types, including new eggs in vitro, particularly when derived from patients with premature ovarian failure or aging and postmenopausal ovaries. Our observations also indicate that there are substantial differences in follicular renewal between adult human and rat ovaries. As part of this chapter, we present in detail protocols utilized to analyze oogenesis in humans and to study interspecies differences when compared to the ovaries of rat females.

Metastatic melanoma of the uterine cervix diagnosed on cervical Pap smear: Case report and literature review.

Metastatic cancer involving the uterine cervix is exceedingly rare, and accounts for less than 1% of cancer deaths. The cervix is an uncommon location for metastatic lesions due to its limited blood supply and fibrous stroma and metastatic melanoma of the cervix is particularly infrequent. To the best of our knowledge and literature review, there have only been nine reported cases in the literature of metastatic melanoma involving the uterine cervix that were diagnosed via cervicovaginal Pap smears, including the case being reported in this paper. Diagnosing metastatic melanoma on cervicovaginal cytology specimens is challenging, not only because of its rarity, but also because of the inherent ability of melanoma to take on many different cytomorphologic appearances. In such cases, the differential diagnosis may include a high-grade squamous intraepithelial lesion, atypical glandular cells, adenocarcinoma and other poorly differentiated malignancies. We report a case of malignant melanoma to the cervix diagnosed by a routine cervical Pap smear in a young woman who was diagnosed with cutaneous melanoma 3 years prior. Because of the diagnosis rendered on her cervical Pap smear, she was subsequently found to have widespread metastatic disease. Although the cervical Pap smear is primarily intended to screen for squamous intraepithelial lesions, a high index of suspicion must be maintained for other less common entities, particularly when there is no evidence of a squamous intraepithelial lesion.

Abnormal expression of p27kip1 protein in levator ani muscle of aging women with pelvic floor disorders - a relationship to the cellular differentiation and degeneration.

BACKGROUND: Pelvic floor disorders affect almost 50% of aging women. An important role in the pelvic floor support belongs to the levator ani muscle. The p27/kip1 (p27) protein, multifunctional cyclin-dependent kinase inhibitor, shows changing expression in differentiating skeletal muscle cells during development, and relatively high levels of p27 RNA were detected in the normal human skeletal muscles. METHODS: Biopsy samples of levator ani muscle were obtained from 22 symptomatic patients with stress urinary incontinence, pelvic organ prolapse, and overlaps (age range 38-74), and nine asymptomatic women (age 31-49). Cryostat sections were investigated for p27 protein expression and type I (slow twitch) and type II (fast twitch) fibers. RESULTS: All fibers exhibited strong plasma membrane (and nuclear) p27 protein expression. cytoplasmic p27 expression was virtually absent in asymptomatic women. In perimenopausal symptomatic patients (ages 38-55), muscle fibers showed hypertrophy and moderate cytoplasmic p27 staining accompanied by diminution of type II fibers. Older symptomatic patients (ages 57-74) showed cytoplasmic p27 overexpression accompanied by shrinking, cytoplasmic vacuolization and fragmentation of muscle cells. The plasma membrane and cytoplasmic p27 expression was not unique to the muscle cells. Under certain circumstances, it was also detected in other cell types (epithelium of ectocervix and luteal cells). CONCLUSIONS: This is the first report on the unusual (plasma membrane and cytoplasmic) expression of p27 protein in normal and abnormal human striated muscle cells in vivo. Our data indicate that pelvic floor disorders are in perimenopausal patients associated with an appearance of moderate cytoplasmic p27 expression, accompanying hypertrophy and transition of type II into type I fibers. The patients in advanced postmenopause show shrinking and fragmentation of muscle fibers associated with strong cytoplasmic p27 expression.