Steffen Blottner

Seasonal timing of sperm production in roe deer: interrelationship among changes in ejaculate parameters, morphology and function of testis and accessory glands.

Roe deer are seasonal breeders with a short rutting season from mid-July to mid-August. The seasonality of reproductive activity in males is associated with cyclic changes between growth and involution of both testes and the accessory sex glands. This study characterizes morphological and functional parameters of these organs prior to, during and after breeding season in live adult roe deer bucks. Size and morphology of the reproductive tract was monitored monthly by transcutaneous (testes, epididymis) and transrectal (accessory glands) ultrasonography. Semen was collected by electroejaculation. Concentration, motility and morphological integrity of spermatozoa as well as the content of proteins and testosterone in semen plasma were evaluated. Proportions of haploid, diploid and tetraploid cells were estimated by flow cytometry in testicular tissue biopsies. Serum testosterone was measured by enzyme immunoassay. Most parts of the male reproductive tract showed distinct circannual changes in size and texture. These changes were most pronounced in the testes, seminal vesicles, and prostate. All reproductive organs were highly developed during the rut only. The volume of ejaculates, total sperm number and percentages of motile and intact spermatozoa also showed a maximum during this period and corresponded with high proportions of haploid cells in the testis. The highest percentages of tetraploid cells were found in the prerutting period. The production of motile and intact spermatozoa correlated with both the protein content of semen plasma and the concentration of testosterone in semen plasma and blood serum. These results suggest the importance of combined actions of the testes and accessory sex glands and the crucial role of testosterone in facilitating the optimal timing of intensified semen production to ensure sufficient numbers of normal spermatozoa in seasonal breeders.

Seasonal Changes of Spermatogonial Proliferation in Roe Deer, Demonstrated by Flow Cytometric Analysis of c-kit Receptor, in Relation to Follicle-Stimulating Hormone, Luteinizing Hormone, and Testosterone

Abstract The roe deer (Capreolus capreolus) is a seasonal breeder. The cyclic changes between totally arrested and highly activated spermatogenesis offer an ideal model to study basic mechanisms of spermatogenesis. In this study, we demonstrated, to our knowledge for the first time, c-kit receptor-positive cells in the testis of roe deer. They were immunohistologically identified mainly as spermatogonia. Analysis of the amount of those cells by flow cytometry shows a distinct seasonal pattern, with pronounced differences between cells in the diploid state and in the G2/M phase of mitosis. The specific seasonal pattern of spermatogonial proliferation results in the increased relative abundance of spermatogonia as well as in their increased total number per testis in November and December. This suggests that cell divisions continue on a level sufficient to accumulate spermatogonia during winter. The serum concentrations of LH and FSH showed a peak in spring; testosterone showed a maximum concentration during the rut (July/August). The peak of both gonadotropins seems to precede the period of stimulated spermatogonial proliferation in spring. The testosterone peak coincides with maximal meiotic intensity in August. The results suggest the importance of testosterone for sperm production, and they provide a basis for detailed investigations of regulatory factors of the proliferation of spermatogonia.

Seasonal Expression of INSL3 and Lgr8/Insl3 Receptor Transcripts Indicates Variable Differentiation of Leydig Cells in the Roe Deer Testis

Abstract Roe deer are seasonal breeders and show cyclic variation in testicular volume and cellular differentiation within the tubular and interstitial testis compartment. We have employed the roe deer as a model to elucidate the expression of the postpubertal Leydig cell marker INSL3 during seasonal changes in Leydig cell differentiation. Roe deer testis and serum samples were collected bimonthly throughout the complete reproductive cycle. Peak levels of testicular Insl3 mRNA and INSL3 immunoprotein were detected well before the onset of rut in April and coincided with the highest percentage of INSL3-positive cell number/square millimeter of testicular interstitial area. During the winter (December, February), roe deer INSL3 was exclusively detected in a subpopulation of alpha-actin-negative, spindle-shaped peritubular cells. Concordant with the increase in INSL3 production in April and 1 mo after the reported LH peak, a sharp increase in serum testosterone concentrations was observed. High serum testosterone concentrations coincided with the presence of detectable 17alpha-hydroxylase, mRNA and protein, in Leydig cells. Upregulation of INSL3 production in spring appeared to reflect LH-dependent differentiation of Leydig cells. The considerable changes in percentage of INSL3 immunopositive cells within the numerically constant interstitial cell population indicated cyclic seasonal de- and redifferentiation of Leydig cells. A complex functional role of the INSL3/LGR8 ligand-receptor system in the roe deer testis was suggested by the detection of specific hybridization signals for roe deer Lgr8 transcripts in Sertoli cells of the roe deer testis.

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.

Split-sample comparison of directional and liquid nitrogen vapour freezing method on post-thaw semen quality in white rhinoceroses (Ceratotherium simum simum and Ceratotherium simum cottoni)

To increase the quality of cryopreserved sperm in white rhinoceros, the liquid nitrogen vapour (LN vapour) freezing and the multi-thermal gradient directional freezing methods were compared. Sixteen white rhinoceros (Ceratotherium simum sp.) were electro-ejaculated. Semen samples were diluted with cryoextender (Tris, lactose, egg-yolk, DMSO) and aliquoted into straws for LN vapour freezing, and glass hollow tubes for directional freezing. The sperm quality was evaluated before and after freezing by assessing the following parameters: motility, morphologic state, acrosomal integrity and plasma membrane function and integrity (i.e. sperm viability) as defined by the hypo-osmotic swelling. Directional freezing improved the sperm viability by 5.6% (p<0.005), progressive motility score by 34.7% and sperm motility index (SMI) by 8.1% (p<0.005) versus LN vapour freezing. When data was categorized into groups of low (<19%), moderate (20-39%) and high (>40%) percentages of morphologically normal, directional freezing (DF) resulted in 31.4% less abnormal acrosomes for the low quality group as well as 18.7% increase in intact acrosomes and 10.9% increase in motility for the high quality group compared to LN vapour freezing (LN) (p<0.01, p<0.03, p<0.01, respectively). LN showed a significant reduction in sperm head volume (5.7%, p<0.05) compared to the prefreeze; whereas, no significant reduction in head volume was demonstrated after DF. Several additives (xanthenuric acid, cytochalasin D, potassium, EDTA) to the basic cryoextender provided no significant improvement in spermatozoal survival after directional freezing. In conclusion, directional freezing proved to facilitate higher gamete survival compared to LN vapour freezing. This is especially effective in ejaculates of low sperm quality and is important in endangered species where high quality semen donors are often not accessible. These results suggest that directional freezing could be valuable particularly for species with limited freezability of spermatozoa.

Characteristic oxidation products of choline plasmalogens are detectable in cattle and roe deer spermatozoa by MALDI-TOF mass spectrometry.

Plasmalogens (1-O-alk-1′-enyl-2-acyl-sn-glycero-3-phosphocholines and -phosphoethanolamines) are important constituents of spermatozoa membranes and possess significant antioxidative properties. This particularly holds as plasmalogens from spermatozoa also possess a very high content of highly unsaturated fatty acyl residues (especially 22:6). The organic spermatozoa extracts of two different ruminants (cattle and roe deer) were analyzed for their contents of characteristic choline plasmalogen oxidation products by matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry. It will be shown that 1-hydroxy-2-docosahexaenoyl-sn-glycero-3-phosphocholine (LPC 22:6) and formyl-LPC 22:6 are reliable measures of lipid oxidation of spermatozoa and allow, accordingly, conclusions about the storage conditions. All data on spermatozoa were also confirmed by the investigation of the oxidation behavior of selected reference compounds. It will be shown that, equally if plasmalogens or diacyl PC species are used, oxidation takes place primarily at the double bond next to the glycerol backbone. These data were additionally confirmed by recording the corresponding post source decay (PSD) fragment ion spectra.

Estrogens are involved in seasonal regulation of spermatogenesis and sperm maturation in roe deer (Capreolus capreolus)

Roe deer (a seasonal breeder, rut: July to August) is a well characterized model for studying the seasonal regulation of testicular activity. However, not much is known about the impact of estrogens on seasonally determined sperm production. We therefore explored the time and cell type specific expression of estrogen receptors and of enzymes involved in steroid biosynthesis in roe deer testicular parenchyma and in the epididymis. Every second month during the entire seasonal cycle five roe bucks were castrated (n=30). Estrogen receptor (ER) alpha, ERbeta and the enzymes P450Aromatase and P450C17 were localized immunohistochemically. The expression levels of ERalpha, ERbeta and P450Aromatase were evaluated by semi-quantitative Western blot. Contrary to the enzyme required for androgen production (P450C17), which is expectedly located only in the Leydig cells and shows an expression increase towards rutting season, a seasonal expression difference of the enzyme required for the conversion into oestradiol (P450Aromatase) is visible only in the epididymis. In the testis, ERalpha expression shows a striking dependency on tubular cell composition, and the single cell expression activity increases towards rut. This implicates that estrogens are directly involved in the regulation of spermatogenesis in the roe buck. In the epididymis, expression of ERalpha is seasonally determined particularly in the ductuli efferentes. ERbeta was detected throughout the year with no distinct dependency on season or the stages of germinative epithelium cycle. We conclude that estrogens in the roe buck influence the seasonally determined sperm production predominantly by the regulated expression of ERalpha.

Apoptosis is not the cause of seasonal testicular involution in roe deer

Apoptosis is involved in the regulation of spermatogenesis. The involution of testes in seasonal breeders might be expected to involve enhanced apoptotic cell elimination. We have compared seasonally changing testicular apoptosis in roe deer with that in non-seasonally breeding cattle. Apoptotic cells were detected as TUNEL-positive cells by both flow-cytometric analysis and in situ localisation of fragmented DNA in tissue sections. Apoptosis-induced DNA fragments were also assessed by enzyme-linked immunosorbent assay (ELISA) in homogenised testicular parenchyma. As expected, the testis mass and the percentage of haploid cells in roe deer showed a seasonal pattern with a significant maximum during the rut (August), whereas no annual variation of these parameters was found in bulls. All three methods for determining apoptosis showed similar findings. Roe deer exhibited significant seasonal fluctuation of total apoptotic activity (ELISA, apoptotic cells per tubule cross section) with a maximum during the breeding season. However, the seasonal differences in the number of apoptotic cells corresponded to the variable total numbers of spermatogonia and spermatocytes per tubule cross section. Thus, the percentages of TUNEL-positive cells related to the combined number of both germ cell types showed no seasonal variance, as confirmed by percentages of apoptotic cells analysed flow-cytometrically. The maximum level of apoptosis during the rut in roe deer was similar to the values obtained during the invariably high spermatogenic activity in cattle. These results suggest that, in roe deer, apoptosis is not the cause of the seasonal involution of testes.

Detection of growth factors in the testis of roe deer (Capreolus capreolus).

Roe deer is a seasonal breeder characterised by a short rutting season in summer. Mature males show synchronised cycles of testicular involution and recrudescence. Therefore, this species is a valuable model to study seasonal regulation of spermatogenesis in ruminants. It is hypothesised that a time-dependent production of testicular growth factors is required to regulate seasonal changes in testis growth and spermatogenesis. To identify potential candidates, total RNA from roe deer testis tissue was extracted at three different seasonal periods (April, August, December), and using RT-PCR the presence of several growth factors (aFGF, bFGF, IGF-I, IGF-II, TGF-alpha, TGF-beta1, TGF-beta3 and two isoforms of VEGF) was detected. Sequencing of the growth factor PCR fragments revealed a high sequence homology between cattle and roe deer. To further explore the expression patterns of the identified growth factors in roe deer their expression levels were standardised using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression. The study demonstrates the expression of several growth factors in roe deer testis and supports the assumption of their seasonally diverse regulation. These results provide the basis to investigate the role of growth factors in the regulation of circannual changes of testicular activity.

Artificial insemination of captive European brown hares (Lepus europaeus PALLAS, 1778) with fresh and cryopreserved semen derived from free-ranging males

This study aimed to establish artificial insemination (AI) protocols to predictably initiate pregnancy during the breeding season in the European brown hare (EBH) (Lepus europaeus PALLAS, 1778). Semen was collected from seven captive and eight free-ranging males by means of electroejaculation. Semen from the free-ranging males was cryopreserved using directional freezing. Total motility/integrity of fresh and frozen-thawed semen was 91.6%/87.7% and 46.9%/53.8%, respectively. Ovulation was induced in ultrasonographically preselected females using a gonadotropin-releasing hormone analogue. Each female was inseminated with 1 mL fresh (Group A, n=16) or frozen-thawed semen (Group B, n=9) at a concentration of 100x10(6) spermatozoa/mL. The use of ultrasonography (10 to 22 MHz) confirmed the intracervical semen deposit, the success of artificial ovulation induction (formation of postovulatory corpus luteum), and permitted the monitoring of individual pregnancies. Although sperm motility/integrity was significantly different between groups, no significant difference was detected in conception rates (A, 87.50%; B, 77.78%). Because of embryonic resorption, there was a slight difference in fertility rate between groups (A, 62.5%; B, 77.78%). Overall, AI in captive EBH using fresh and frozen-thawed semen achieved successful fertility rates. Long-term cryopreserved semen was used to bring new genetic material from the wild into a genetically limited captive population without extensive animal transport. Therefore, AI has the potential to enhance breeding programs for EBH especially when cryopreserved semen from wild donors is used.