Katharina Spanel-Borowski

Reduction in corpora lutea number in obese melanocortin-4-receptor-deficient mice

Obese melanocortin-4-receptor-deficient (MC4R-/-) male mice are reported to have erectile dysfunction, while homozygous MC4R-/- female mice are apparently fertile. A recently established obese mouse strain, carrying an inactivating mutation in the MC4R gene, revealed difficulties in breeding for the homozygous female mice. This prompted us to determine the presence of follicles and corpora lutea (CL) in ovaries of MC4R-/- mice aged 3–6 months in comparison to wild type (MC4R+/+) littermates. Serial sections of formaldehyde-fixed ovaries of mice with vaginal signs of estrus and metestrus were assessed for the number of healthy and regressing follicles and CL. The number of CL, as an estimate for the ovulation rate, decreased to zero during aging in MC4R-/- mice. The number of small- (diameter 100–200 micrometer) and large-sized follicles namely antral follicles (diameter >200 micrometer) were slightly increased in MC4R-/- compared to MC4R+/+ mice. Greater differences were found in very large to cystic follicles, which were more numerous in MC4R-/- mice. The number of regressing antral follicles was higher in the MC4R-/- group compared to the MC4R+/+ group. This was associated with a wide range in the number of collapsed zonae pellucidae as the last remnants of regressed follicles. A conspicuous hypertrophy of the interstitial cells was noted in 6-month-old MC4R-/- mice. In conclusion, cystic follicles and the reduction in CL number point to a decreased ovulation rate in obese MC4R-/- mice.

Expression of KIT in the ovary, and the role of somatic precursor cells.

KIT is a type III receptor protein tyrosine kinase, and KITL its cognate ligand. KIT can mediate its effects via several intracellular signalling pathways, or by formation of a cell-cell anchor with its ligand. Through these mechanisms, KIT controls fundamental cellular processes, including migration, proliferation, differentiation and survival. These cellular processes are modulated by soluble KIT, a cleavage product of KIT, generated at the cell membrane. A cell-retained KIT cleavage fragment also arises from this cleavage event. This cleavage fragment must be distinguished from truncated KIT (trKIT), which originates through cryptic promoter usage. The expression of trKIT is highly restricted to postmeiotic germ cells in the testis. In contrast, KIT, together with its cleavage products, is present in somatic cells and germ cells in the gonads of both sexes. A functional KITL/KIT system is mandatory for normal population of the gonads by germ cells. Signalling via the KITL/KIT system promotes the growth, maturation, and survival of germ cells within the gonads, and prevents meiotic entry and progression. In addition to its importance in germ cell biology, the KITL/KIT system is crucial for gonadal stromal differentiation. During foetal life, KIT is expressed by testicular stromal precursor cells, which develop into Leydig cells. In the ovary, stromal cell KIT expression accompanies theca layer development around advanced follicles. After ovulation, KIT-immunopositive cells translocate from the theca layer to the luteal ganulosa where they contribute to a delicate cellular network that extends between the fully luteinised large luteal cells. In the outer regions of the developing corpus luteum, a highly conspicuous subpopulation of KIT/CD14-double-immunopositive cells can be observed. KIT/CD14-double-immunopositive cells are also seen in the haematopoietic-like colonies of long-term granulosa cultures established from late antral follicles. These cultures demonstrate expression of pluripotency marker genes such as octamer binding transcription factor-3/4 and sex determining region Y-box 2. The KIT/CD14-double-immunopositive cells can be purified and enriched by KIT-immunopositive magnetic cell sorting. Subsequent exposure of the KIT-expressing cells to the hanging drop culture method, combined with haematopoietic differentiation medium, provides the signals necessary for their differentiation into endothelial and steroidogenic cells. This suggests that monocyte-derived multipotent cells are involved in ovarian tissue remodelling. In summary, multicelluar KITL/KIT signalling organizes the stroma in the ovary and testis; monocyte-derived multipotent cells may be involved.

The sacrotuberous and the sacrospinous ligament--a virtual reconstruction.

Little is known about the morphometric properties of the sacrotuberous ligament (ST) and the sacrospinous ligament (SS). The influence of ligaments on pelvic stability and the extent of reconstruction in case of instability are controversially discussed. The ST and the SS of 55 human subjects fixed in alcohol solution and of four fresh cadavers were measured. Both ligaments were defined as geometric figures. The ST was a contorted bifrustum, while the SS was a contorted frustum, both with elliptic planes. In all cases investigated, the ST and the SS fibres were twisted. For men, the ST and the SS had a mean length of 64 and 38 mm. For women, lengths of 70 and 46 mm were measured in the ST and the SS. The ST length, height and cross-sectional area showed gender-specific differences at statistically significant level. The ST and the SS volumes correlated closely, regardless of gender or side. Measurements of fresh ligaments of four unfixed cadavers showed similar results. The data obtained were then used to generate computer-based three-dimensional models of both ligaments, using the Catia software. Conclusively, the virtually generated ST and SS are suitable models to be included in pelvic fracture simulation, using the finite element method.

Correlation between expression of selectins and migration of eosinophils into the bovine ovary during the periovulatory period

Abstract. Leukocytes enter specific ovarian areas at a precisely defined moment, influencing cyclically changing structures such as follicles and corpora lutea. As yet, no studies have been published on the trafficking mechanisms involving the interaction between adhesion molecules on endothelial cells (ECs) and those on leukocytes. First, antibodies against human adhesion molecules were examined by flow cytometry with the aim of identifying the same bovine antigen. Western blot analysis and immunoprecipitation revealed that the molecules had the same molecular weight as their human counterparts. Afterwards, we investigated the distribution of these antigens in various ovarian stages using immunohistology. Among the molecules, P-selectin (CD62P) and L-selectin (CD62L) showed stage-dependent expression, and were thus examined further. In the preovulatory follicle, microvascular ECs were negative for CD62P. Few of the leukocytes expressed CD62L. In a freshly ruptured follicle, CD62P expression was found in the dilated vessels of the former thecal layer. Simultaneously, a large proportion of the rapidly increased numbers of leukocytes, mainly eosinophils, located around the microvessels of the outer thecal layer expressed CD62L. In the early corpus luteum development stage, CD62L showed peak expression with 70%–80% positive cells compared to leukocytes. In the secretory stage, the septal venules showed a consistent, but now weak, staining for CD62P. Few leukocytes expressed CD62L. During regression, the total number of leukocytes, now representing macrophages, increased significantly, but the proportion of CD62L-positive cells remained constant. In summary, we found a strong correlation of CD62P expression on activated ECs and the appearance of CD62L-positive leukocytes in the early corpus luteum development stage, suggesting the participation of both selectins in the migration of eosinophils under physiological conditions.

Description of the iliolumbar ligament for computer-assisted reconstruction.

STUDY DESIGNnThe iliolumbar ligament (IL) was examined using morphometric and virtual methods.nnnOBJECTIVESnA macroscopic study was performed to measure the anterior (AIL) and the posterior part of the IL (PIL).nnnSUMMARY OF BACKGROUND DATAnThough being a widely accepted cause of low back pain and lumbosacral instability, the IL is neglected in computer-based biomechanical studies due to the lack of morphometric information.nnnMETHODSnFrozen sections prepared from 29 human subjects were measured and 7-tesla MR images made to distinguish the AIL and PIL. Cuboids were designated as geometric figures to both parts of the ligament, allowing computer-based calculations of length, surface, volume and angle of positional relationships.nnnRESULTSnBased on 7-tesla MR imaging, virtual reconstruction was conducted for one male pelvis, including the IL. While left- and right-side parameters varied at a statistically significant level, no gender-dependencies could be determined. Lengths of 30 and 25 mm were measured for the AIL and PIL, as well as heights of 17-19 mm, respectively, and a thickness of 4mm.nnnCONCLUSIONSnCorrelations between the side-dependent parameters and the AIL and the PIL of the same side indicate close functional relationships. Additional dependencies suggest that the IL is capable of compensating age-related as well as bone-attributed alterations in lumbosacral morphology. The IL data and the visualised ligament structures contribute to determination of the influence of the IL in spinal and sacroiliac stability by means of computer-assisted biomechanics.

Anatomy of the human thoracolumbar Rami dorsales nervi spinalis

Medial, lateral, and intermedial ramifications have been described for the dorsal branch of the human spinal nerve (R. dorsalis n. spinalis, (RDNS)). Further branching has not been described. We report a ventral approach for dissecting the nerves around the thoracolumbar vertebral column to visualise the spreading of the nerves within the dorsal muscles and towards the skin. We defined three compartments of the deep back muscles in the thoracolumbar region: (A) the origin from the (1) transverse, (2) accessory, and (3) mammillary processes in the lumbar segments, (B) from the (1) ribs, (2)transverse and, (3) articular processes in the thoracolumbar segments. Each compartment was supplied by a ramification of the RDNS. The medial muscle compartment was reached by the descending medial branch of the RDNS. The lateral iliocostal compartment was innervated by an ascending lateral branch of the RDNS, and also by the descending distal branches of an intermedial branch of RDNS. This is a long nerve of the intermedial branch of the RDNS extended to the dorsal-caudal area, where the lateral and the intermedial nerve connected. This nerve, termed as the dorsal intermedial branch of the RDNS, innervated the skin in a more caudal region. Such nerve divided the lateral and the intermediate compartments. A short intermedial branch entered the intermediate segmental compartment from the ventral side. This is a ventral intermedial branch of the RDNS. The dorsal branches were often connected by a connecting branch of the RDNS. The lateral compartment represented the Iliocostalis. The medial and intermediate compartments comprised the Longissimus, part of the Iliocostalis, and additional dorsal muscles.

Development and regression of non-capillary vessels in the bovine corpus luteum

Abstract. The corpus luteum life cycle is accompanied by capillary growth, maturation and degeneration. Arterial blood vessels are thought to undergo hyperplasia and hypertrophy during the stage of regression, as is the case with non-capillary vessels. In this study, we used morphological studies to show that the development of non-capillary vessels occurs at other corpus luteum stages. Non-capillary vessels were present at the developmental stage of the corpus luteum, and increased markedly in number in the subsequent stages. After double-staining for ASM-1 actin and Ki-67 nuclear antigen, the proliferation of smooth muscle cells (SMCs) was only detected during stages of development and secretion. When the capillaries had disappeared at the regression stage, the arterial blood-vessel walls thickened noticeably. This was attributed to the development of fibroelastosis as shown by staining for collagenous and elastic fibres. In conclusion, the bovine corpus luteum represents a physiological model for studying arteriolization at all stages of development and secretion. At the regression stage, arterioregression sets in.

Limitation of microwave treatment for double immunolabelling with antibodies of the same species and isotype

Abstract. Microwave treatment (MW) involves completely blocking contaminating staining in the double-labelling technique, using primary monoclonal antibodies from the same species and the same isotype as well as the same secondary antibody (ab). However, we noticed some limitations when locating proliferating cell types in cryostat and paraffin sections using the advantages presented by MW. Control experiments have shown that MW does not diminish contaminating staining when cytoplasmic (desmin, ASM-1) or nuclear (Ki-67) antigens have been labelled with antibodies in the first round of immunolabelling. In contrast to the cell surface antigen, CD18, where the primary ab had to be crosslinked by a secondary ab to obtain contaminating staining, this was observed for the detection of cytoplasmic or nuclear antigens only labelled with a primary ab. In conclusion, for double immunolabelling with abs from the same species and the same isotype, MW is not able to completely abolish contaminating staining.

Demonstration of pelvic anatomy by modified midline transection that maintains intact internal pelvic organs

Gross dissection for demonstrating anatomy of the human pelvis has traditionally involved one of two approaches, each with advantages and disadvantages. Classic hemisection in the median plane through the pelvic ring transects the visceral organs but maintains two symmetric pelvic halves. An alternative paramedial transection compromises one side of the bony pelvis but leaves the internal organs intact. The authors propose a modified technique that combines advantages of both classical dissections. This novel approach involves dividing the pubic symphysis and sacrum in the median plane after shifting all internal organs to one side. The hemipelvis without internal organs is immediately available for further dissection of the lower limb. The hemipelvis with intact internal organs is ideal for showing the complex spatial relationships of the pelvic organs and vessels relative to the intact pelvic floor. Anat Sci Educ 3:254–260, 2010.