Katarina Jewgenow

Apoptosis within bovine follicular cells and its effect on oocyte development during in vitro maturation

Developmental competence of oocytes is compromised if they originate from atretic follicles. Apoptosis is the underlying process of atresia. Apoptotic changes in follicular cells are thought to influence the outcome of IVF. The aim of this study was to investigate apoptosis in different compartments of single bovine follicles (follicular wall, granulosa and cumulus cells (CC)) in relation to COC morphology, and to determine whether the addition, in vitro, of exogenous follicular cells from atretic follicles to maturing cumulus oocyte complexes (COCs) influenced the development of oocytes. Antral follicles were dissected from bovine ovaries and opened to obtain COCs and free floating granulosa cells (GC). The COCs were classified according to morphology. Apoptosis was determined in cumulus and granulosa cells and in homogenates of the remaining follicular wall. For every morphological class of COCs, a large variability of apoptotic expression was found in all follicle compartments. Follicular wall apoptosis was not correlated to COC morphology or to the percentage of apoptotic granulosa or cumulus cells. In grade 1 (best morphology) COCs, the degree of apoptosis in granulosa cells was comparable to cumulus cell apoptosis (P<0.01). The overall expression of apoptosis in granulosa cells of follicles containing grade 3 COCs (median+/-median absolute deviation: 37.8+/-13.8%) was significantly higher (P<0.05) than in follicles with grade 1 (22.7+/-10.4%) or grade 2 COCs (20.0+/-17.0%). About 48.3% of grade 3 COCs possessed strongly apoptotic cumulus cells compared to 27.8 and 28.2% of grade 1 or grade 2 COCs, respectively. Nonapoptotic cumulus complexes were observed in grades 1 and 2 COCs only. Adding exogenous follicular cells from atretic follicles to bovine COCs (grades 1 and 2) during in vitro maturation (IVM) had no impact on fertilization, blastocyst formation or hatching after IVF. This is of particular practical relevance to embryo production after ovum pick up (OPU), as during this process, good quality COCs are cultured together with simultaneously collected slightly atretic COCs.

Ultrasonography as an important tool for the development and application of reproductive technologies in non-domestic species.

Ultrasound imaging in reproductive sciences offers new opportunities regarding optimization of the induction of the sexual cycle and ovulation, superovulation regimes, contraception programs, semen collection and testicular sperm extraction techniques, ovum pick up and ovarian transplantation procedures, as well as the application of artificial insemination, embryo collection and transfer. In non-domestic species, most of which lack basic data, ultrasonography is an ideal tool to study reproductive biology in both captive and wild populations. The use of this imaging modality led us to develop new, or modify established, reproductive technologies. Ultrasonography has been an integral part of over 200 assisted reproduction procedures in 17 mammalian species performed by our research team between 1992 and 1999. These procedures included the initial characterization of sexual cycles, hormonal cycle induction, semen collection by electroejaculation or manual stimulation, non-surgical artificial insemination (AI), non-surgical embryo transfer and temporary hormonal contraception. For these investigations, a variety of newly developed equipment was applied and species-specific hormonal treatments designed. We used several commercial and customized ultrasound systems with a variety of technical features. Some relevant improvements of these applications will be described and the role of ultrasonography elucidated to.

The recovery of preantral follicles from ovaries of domestic cats and their characterisation before and after culture

The mechanical dissection of ovaries from domestic cats provided high numbers of preantral follicles (300–24 500 per ovary) with diameters of 40–90 μm. The high variability between the individual queens was not correlated to age, cycle, breed or season. The number of primordial and primary follicles was significantly dependent upon the total recovery rate (r = 0.997; P < 0.0001). The growing population (secondary follicles) was relatively constant until the isolation rate dropped below 1000 per ovary. Then the number of growing follicles was correlated to the resting pool. Follicle viability, as estimated by trypan blue staining, dropped immediately after isolation to 32.5%. After 1 week of culture, follicle viability varied depending on the method of culture (in monolayer, 33.3%; co-culture with granulosa cells, 37.5%; in collagen gels, 30.8%; in conditioned medium, 9.3%; on agarose pad, 17.9%). A 10μm increase in diameter was observed in approximately 65% of growing follicles (61.3–68.6% depending on growth conditions). The number of surrounding granulosa cells decreased and 32% of the oocytes were without granulosa cells. The addition of epidermal growth factor (EGF) and insulin-transferrin-selenite (ITS) to the culture medium prevented these alterations in structure. In the control group only 22% of intrafollicular oocytes had an intact germinal vesicle as opposed to ITS-medium (57%) and EGF-medium (33%). Cryopreservation of preantral follicles resulted in a 60% reduction in follicle viability.

Isolation of preantral follicles from nondomestic cats—viability and ultrastructural investigations

Abstract Large scale isolation of small preantral follicles (40–90 μm) from nondomestic cats (lion, puma, cheetah, jaguar, and three kinds of tigers) is described and compared with domestic cats. The viability of preantral follicles was estimated by trypan blue staining of granulosa cells and/or by 5-bromo-2′-deoxy-uridine (BrdU) incorporation into oocytes and granulosa cells during short term culture. Native and isolated preantral follicles were compared ultrastructurally. In nondomestic cats the mechanical dissection of ovaries provided 0–12 500 follicles per ovary with a viability of 20–50%, estimated by trypan blue staining. Even the follicles recovered from domestic cats, whose ovaries are considerable smaller than ovaries from all other felids, are characterised by only 28.7% viable follicles. The follicles from one Siberian tiger and three Indian lions were cultivated and their in vitro viability assessed by BrdU labelling. Lion follicles were comparable to domestic cat follicles with respect to BrdU incorporation. Tiger follicles were characterised by a decreased staining of granulosa cells. The ultrastructure of feline oocytes appears similar to that of most mammalian species and was only slightly affected during the isolation procedure. A central vesicular body was only observed in tiger oocytes.

Short-term preservation of canine preantral follicles: effects of temperature, medium and time.

The use of the large pool of preantral follicles is a promising alternative to provide high numbers of fertilizable oocytes to reproductive biotechnology. This issue is particularly important to canids, since current rates of success of in vitro techniques using oocytes are very limited, and many species within this family are threatened by extinction. The aim of this study was to evaluate effects of temperature, medium and time on morphology and viability of canine preantral follicles during short-term preservation. Canine ovaries were cut into fragments which were incubated in 0.9% NaCl solution or in minimum essential medium (MEM) at 4, 20 or 38 degrees C for 2, 6, 12 or 24 h. Afterwards, preantral follicles were analyzed by histology, transmission electron microscopy and viability testing using trypan blue, calcein-AM and ethidium homodimer-1. Percentages of morphological normal and viable follicles were maintained similar to control (time 0 h) after incubation in 0.9% NaCl at 4 or 20 degrees C for up to 6h and at 38 degrees C for 2 h. Using MEM, such preservation was possible for 12h at 4 or 20 degrees C, and for 6h at 38 degrees C. These results indicate that preservation of canine preantral follicles might be better accomplished through hypothermic (4 or 20 degrees C) storage in MEM, which ensures maintenance of morphology and viability for up to 12h.

Cryobanking of viable biomaterials: implementation of new strategies for conservation purposes

Cryobanking, the freezing of biological specimens to maintain their integrity for a variety of anticipated and unanticipated uses, offers unique opportunities to advance the basic knowledge of biological systems and their evolution. Notably, cryobanking provides a crucial opportunity to support conservation efforts for endangered species. Historically, cryobanking has been developed mostly in response to human economic and medical needs — these needs must now be extended to biodiversity conservation. Reproduction technologies utilizing cryobanked gametes, embryos and somatic cells are already vital components of endangered species recovery efforts. Advances in modern biological research (e.g. stem cell research, genomics and proteomics) are already drawing heavily on cryobanked specimens, and future needs are anticipated to be immense. The challenges of developing and applying cryobanking for a broader diversity of species were addressed at an international conference held at Trier University (Germany) in June 2008. However, the magnitude of the potential benefits of cryobanking stood in stark contrast to the lack of substantial resources available for this area of strategic interest for biological science — and society at large. The meeting at Trier established a foundation for a strong global incentive to cryobank threatened species. The establishment of an Amphibian Ark cryobanking programme offers the first opportunity for global cooperation to achieve the cryobanking of the threatened species from an entire vertebrate class.

Quantity rather than quality in teratospermic males: a histomorphometric and flow cytometric evaluation of spermatogenesis in the domestic cat (Felis catus).

Abstract Teratozoospermia (ejaculation of <40% morphologically normal sperm) commonly occurs within the Felidae, including certain domestic cats, but the cellular and molecular mechanisms that give rise to this phenomenon remain unknown. This study quantified spermatogenesis to identify differential dysfunctions in teratospermic versus normospermic (>60% normal sperm/ejaculate) domestic cats. Sperm used were from electroejaculates and cauda epididymides. Testes from 10 normo- and 10 teratospermic males were obtained by castration and then evaluated by histomorphometry, flow cytometry, and testicular testosterone enzyme immunoassay. Some morphometric traits (tubular diameter, epithelium height, interstitial area, number of Leydig cells, and blood vessels per cross-section) as well as testicular testosterone concentrations were similar between groups, but testicular volume was greater in teratospermic males. Stage frequencies differed also between both cat populations, suggesting possible dysfunctions in spermiation. Quantification of cell populations in most frequent stages revealed more spermatogenic cells and fewer Sertoli cells per tubule cross-section as well as per tissue unit in teratospermic donors. Hence, the ratio of spermatogenic cells per Sertoli cell was elevated in the teratospermic cat. DNA flow cytometry confirmed higher total spermatogenic and meiotic transformations in teratospermic males. In summary, compared with normospermic counterparts, teratospermic cats have a higher sperm output achieved by more sperm-producing tissue, more germ cells per Sertoli cell, and reduced germ cell loss during spermatogenesis. Gains in sperm quantity are produced at the expense of sperm quality.

Non-invasive Monitoring of Hormones: A Tool to Improve Reproduction in Captive Breeding of the Eurasian Lynx

The survival of many critical endangered mammal species is often depending on successful captive breeding programmes which include the future option of reintroduction to the wild. Breeding in captivity also demands the application of modern assisted reproductive techniques to ensure maximal biodiversity, but knowledge on reproductive physiology is often limited. Therefore, non-invasive monitoring of urinary and faecal hormones has become an important tool for reproductive management. To exemplify the importance of non-invasive hormone monitoring, we choose the Eurasian lynx as a model for the worlds most endangered felid species, the Iberian lynx. We analysed faecal samples of pregnant and pseudo-pregnant female Eurasian lynxes during a 3-year study period. Compared to pre-mating levels faecal progesterone metabolite profiles revealed a tendency towards higher levels in pregnant and pseudo-pregnant females with no difference between both categories. Oestrogen levels raised in both pregnant and pseudo-pregnant females with a tendency to be more elevated and prolonged in pregnant females. Surprisingly both E2 and P4 metabolites were highly correlated (r(2) =0.8131, p < 0.0001) showing a postpartum increase both in pregnant and pseudo-pregnant females. The results from the Eurasian lynx revealed that the measurement of faecal progesterone metabolites led to profiles dissimilar to profiles shown in other felid species, but similar to those from faecal gestagen metabolite analysis in the Iberian lynx. To identify faecal gestagen and oestrogen metabolites a radio-metabolism study was performed. Using the progesterone immunoassay two major progesterone metabolites were detected demonstrating that the assay indeed tracks the relevant metabolites. The oestrogen assay measured authentic 17beta-oestradiol and oestrone, and their conjugates. The analysis of the faecal metabolite composition in samples from early and late pregnancy and lactation particularly revealed a distinct shift in the relation between 17beta-oestradiol and oestrone that changed in favour of oestrone. This might indicate different hormone sources during and after pregnancy (corpus luteum, placenta). We hypothesize, that placental steroid analysis in combination with other highly sophisticated analytical techniques, like liquid chromatography mass spectrometry or urinary relaxin analysis may led to analytical options to confirm pregnancy and to differentiate this from pseudo-pregnancy in lynx species.

Embryonic gene activation in in vitro produced embryos of the domestic cat (Felis catus)

Accurate embryonic gene activation (EGA) is essential for the embryos developmental potency and reflects the quality of in vitro produced embryos. To describe the dynamic and temporal patterns of EGA in the cat, the mRNA expression of developmentally important genes (DNA methyltransferases 1 and 3A, DNMT1 and DNMT3A; gap junction protein α 1, GJA1; transcription factor octamer 4, POU5F1 (OCT4); insulin-like growth factor (IGF) 1 and 2 receptors, IGF1R and IGF2R) was examined by RT-PCR techniques in preimplantation embryos obtained after in vitro maturation and IVF. Furthermore, influences of ICSI and sperm cryopreservation on the relative mRNA abundance in 4-5-days-old morulae were analyzed. Total RNA was obtained from immature and matured oocytes, 2-cell embryos, 4-cell embryos, and 8-16-cell embryos, morulae, and blastocysts. RNA was transcribed into single-stranded cDNA by reverse transcriptase. After amplification, a nonfelid standard RNA was used for semiquantitative analysis. Our results showed an increase in transcript abundance from the matured oocyte to the 2-cell embryo for all examined genes except for IGF2R, indicating that, in vitro, the embryonic genome is activated shortly after fertilization. However, the activation pattern varied markedly between the different genes. We also found different patterns of mRNA expression for the examined genes in morulae produced either by IVF or ICSI, and using fresh or cryopreserved sperm. Owing to high variations within the single groups of compared morulae, we were able to observe only a tendency toward higher relative mRNA expression in embryos derived by IVF with fresh sperm in comparison to all other groups.

Non cat-like ovarian cycle in the Eurasian and the Iberian lynx - ultrasonographical and endocrinological analysis.

The Iberian lynx is considered the most endangered felid species. Therefore, an ex situ conservation program was initiated to protect this species from extinction. Additional knowledge on lynx reproduction biology and reliable methods for reproductive monitoring are important for developing a captive breeding program. The aim of this study in lynx was to implement transrectal ultrasonography to visualize ovarian structures (follicles, corpora lutea) and to assess ovarian activity in addition to analysis of serum progesterone and oestradiol. Because of limited access to Iberian lynxes, the less-endangered Eurasian lynx and bobcat were also studied in this comparative study. Recent endocrinological studies based on faecal and urinary progesterone and oestrogen metabolites revealed that steroid profiles in both these species were alike and did not follow the typical pattern of other felids. Pregnancy diagnosis was not possible, since progesterone concentrations did not differ between pregnant and pseudopregnant animals. Progesterone was also detected after parturition as well as after weaning until the onset of a new oestrous cycle. In the present study, the presence of corpora lutea during the non-breeding season was confirmed by ultrasonography and by elevated serum levels of progesterone averaging 3.56 +/- 1.3 ng/ml in Eurasian and 6.1 +/- 0.26 ng/ml in Iberian lynx, respectively. The ultrasonographical findings on the ovarian structures suggest strongly that corpora lutea developed after ovulation stay active until November and regress before the onset of the next oestrus.