F. M. F. Van Dissel-Emiliani

Regulation of spermatogonial proliferation.

In nonprimates the spermatogonial compartment can be subdivided into (morphologically) undifferentiated spermatogonia and differentiating spermatogonia. Each cycle of the seminiferous epithelium the proliferative activity of the undifferentiated spermatogonia is stimulated, probably by factors secreted by Sertoli cells. Subsequently, during a period of active proliferation many Aal spermatogonia are formed. In the normal situation around epithelial stage III, proliferation is inhibited by the differentiating spermatogonia by way of a negative feedback system, probably involving a spermatogonial chalone. Then most of the Aal spermatogonia formed differentiate into Al spermatogonia. For this differentiation vitamin A, or factors secreted by Sertoli cells under the influence of vitamin A, is/are necessary. In the normal situation there is no regulation of the density of the undifferentiated spermatogonia. Different tubular areas can contain widely varying numbers of stem cells and other undifferentiated spermatogonia and consequently can produce widely varying numbers of Al spermatogonia. Only in extreme circumstances, such as after irradiation, the stem cells change the ratio between self-renewing and differentiating divisions in favor of self-renewal. Furthermore, in this situation the proliferation of the undifferentiated spermatogonia is not inhibited at epithelial stage III because of the lack of differentiating spermatogonia. Density regulation does take place during the development of the differentiating spermatogonia. In the chinese hamster it appeared that despite the variation in the numbers of Al spermatogonia produced in different areas, the density of the preleptotene spermatocytes was very much the same. It was found that the even distribution of spermatocytes in the epithelium was achieved by a density-dependent degeneration of differentiating spermatogonia in such a way that many of the latter cells degenerated in high density areas and only few or none in low density areas. In primates the undifferentiated spermatogonia can be subdivided into Ap and Ad spermatogonia. Both Ap and Ad spermatogonia can be seen to be topographically arranged in clones of 1 or 2n cells in situations in which their density is low. The Ad spermatogonia do not proliferate, but after cell loss these cells were found to transform into Ap spermatogonia that start to proliferate. The Ap spermatogonia only divide once every epithelial cycle, renewing themselves and giving rise to B spermatogonia. In the monkey the number of Ap spermatogonia could be increased by FSH treatment. Hence, there may be a correlation between FSH levels and the numbers of Ap spermatogonia. Fu

Survival and proliferation of rat gonocytes in vitro.

Quiescent gonocytes were isolated from fetal testes of rat 18-day post coitum and cultured alone or on monolayers of somatic cells from different origins. The gonocytes specifically adhered to Sertoli cells, isolated from 21 to 23-day-old rat testes; this adherence was necessary for their survival in vitro. Addition of follicle-stimulating hormone and testosterone to these cultures did not increase the viability of the gonocytes. Serum was found to be deleterious to the germ cells. Electron-microscopic examination of Sertoli-cell-gonocyte co-cultures revealed the presence of numerous adhesion plaques between these cells, indicating that Sertoli cells and gonocytes are able to communicate in vitro. Gonocytes, in co-culture with Sertoli cells, were viable for at least 9 days. The gonocytes did not spontaneously resume proliferation. The simple culture system described in the present paper should be useful in studying the nature of the factors that are responsible for sending the quiescent gonocytes into the cell cylce and for stimulating the formation of A spermatogonia, a process characterizing the start of spermatogenesis.

Changes in Water Content, Collagen Degradation, Collagen Content, and Concentration in Repeated Biopsies of the Cervix of Pregnant Cows

Abstract The objective of the present study was to assess if cervical ripeness could be quantified by measuring the percentage of denaturation of the collagen network of the stromal layer. Biopsy specimens from the caudal part of the cervix were obtained from nine pluriparous cows between Days 149 and 157 of gestation (second-trimester biopsy), at exactly Day 275 of gestation (term biopsy), and shortly after calving (calving biopsy). The samples were divided into a superficial stromal part and a deep stromal part. The water content was derived from the weight of the samples before and after lyophilization. A colorimetric assay was used to assess the percentage of collagen denaturation by determining the extinction at 570 nm of hydroxyproline released from α-chymotrypsine-treated samples. By incorporating a hydroxyproline standard series in the measurements, the insoluble collagen content (µg/mg dry wt) as well as the insoluble collagen concentration (µg/mg wet wt) could be derived. The water content of both layers of the cervix significantly increased between midpregnancy and parturition (P < 0.01). The insoluble collagen content and the insoluble collagen concentration were significantly increased at term (P < 0.01 and P < 0.05, respectively) but were significantly decreased at calving (P < 0.05 and P < 0.01, respectively). Both parameters showed no significant differences between the superficial and deep stromal layer, and they were significantly correlated with each other. A significant increase in the percentage denaturation of the deep stromal layer occurred between the second trimester and term pregnancy (P < 0.01), whereas at calving, the percentage denaturation had not significantly increased compared to term. The percentage of collagen denaturation of the superficial stromal layer did not significantly change with stage of gestation or at parturition. Our findings indicate that cervical ripening is a combination of increased collagen synthesis and increased percentage of collagen denaturation, whereas at calving, an increased digestion of the denatured collagen leads to increased collagen loss from the cervical connective tissue. The finding that cervical ripening mainly takes place in the deep stromal layer of the cervix emphasizes the importance of a detailed description of the tissue sampling sites for a proper interpretation of the results obtained from biochemical studies of the cervix.

Heterogeneity in the in vitro survival and proliferation of human seminoma cells

The in vitro culture conditions allowing survival and initial proliferation of murine primordial germ cells from 10.5 days post coitum embryos, which include the use of a murine embryonal fibroblast (STO) feeder, were applied to 21 human seminomas, composed of tumour cells which are considered as the malignant counterparts of human primordial germ cells. Cells from 18 seminomas attached poorly to STO, and only a few survived through day 10. In contrast, three seminomas showed a higher degree of attachment. Two of them showed initial proliferation and enhanced survival: 30 days for tumour SE1 and 25 days for tumour SE3. Tumour SE1 was more extensively studied, using the culture conditions allowing the derivation of pluripotent embryonic stem cells from 8.5 days post coitum murine primordial germ cells, which include the use of STO feeder, stem cell factor, leukaemia inhibitory factor and basic fibroblast growth factor. The presence of stem cell factor was necessary and sufficient for colonies of tumour cells to form during the first 3 days of culture. While the cell number decreased after day 3 in medium without fetal calf serum, it increased until day 9 in medium containing fetal calf serum. No reprogramming of SE1 cells to pluripotent stem cells was observed. Our data indicate that seminomas form a tumour population with a heterogeneous in vitro behaviour not equivalent to that of 8.5-10.5 days post coitum murine primordial germ cells.

Between prepartum luteolysis and onset of expulsion

In the cow the foetal endocrine signals that initiate the calving process result in prepartum luteolysis. Withdrawal of progesterone (P4) action is a prerequisite for a normal calving. The rather abrupt declining influence of P4 is followed by a cascade of physiological processes in the myometrium and cervix. This contribution will focus on some of these events. Like in many other species, the myometrium in cows is not completely inactivated during pregnancy. So-called contractures have been registered during the final weeks of gestation and their EMG-characteristics in cows show a low frequency (on average: 13.6 per day) and long duration (on average 12.1 min). They are not evenly spread over the day because they occur less frequently when the cows are disturbed for feeding or cleaning their stables. Contractures affect several foetal functions. In the cow these contractures disappear during a period of about 8-9h when maternal plasma P4 levels are rapidly declining before calving. There is experimental evidence that this temporary inhibition is associated with prepartal luteal regression. The cause of this inhibition is still unknown. Because nitrous oxide inhibits smooth muscle cells and evidence in laboratory animals indicates that expression of the inducible form of nitrous oxide (iNOS) is downregulated in myometrium, but upregulated in the cervix around the onset of parturition, we started to investigate the role of this enzyme in bovine tissues around calving. By means of a RT-PCR technique, we obtained a first indication that iNOS is hardly expressed in the myometrium during calving, while expression was clearly detected at day 4 after calving. Analysis of prepartum en periparturient biopsies from myometrium and cervix with quantitative PCR is still underway. In six pregnant cows, provided with uterine EMG-electrodes and with ultrasonic crystals implanted on the caudal cervical rim to measure cervical dilatation, calving was induced with an injection of prostaglandin (PG) F2alpha. While maternal plasma P4 levels had significantly declined within 8h after PG treatment, the myometrium escaped from temporary inhibition with the development of a parturient contractility pattern on average at 13.5h after injection. However, it was only at 28 h after PG treatment that the first sustained increase of the opening of the vaginal ostium of the cervix was measured.

Regional Differences in Water Content, Collagen Content, and Collagen Degradation in the Cervix of Nonpregnant Cows

Abstract The cow could be a suitable model for studies concerning functional changes of the cervix. However, as in many species, the bovine cervix becomes softer in texture during the follicular phase of the estrous cycle compared to the luteal phase. In the present study, we explored if changes in the collagen network take place that could be responsible for this phenomenon and if regional differences in water content, collagen content, and collagen degradation along the cross-sectional and longitudinal axes of the cervix were present. Two groups of nonpregnant animals with different progesterone status were studied. One group (n = 11) was under high progesterone influence, and the other group (n = 12) was under low progesterone influence. The water content was derived from the weight of the samples before and after lyophilization. The collagen content and the ratio of collagenous to noncollagenous proteins (hydroxyproline:proline ratio) were determined by performing amino acid analysis on hydrolyzed samples using high-performance liquid chromatography. Collagen denaturation was quantified with a colorimetric assay by determining the amount of hydroxyproline released from samples treated with α-chymotrypsine. The water content of the superficial layer of the submucosa was always significantly (P < 0.01) higher than the water content of the deep layer in the vaginal, mid, and uterine segments, but this was unrelated to the progesterone status of the animals. No effect of the tissue layers or of the progesterone status of the animals on the collagen content was observed, but an effect of segment was noted. The collagen content (µg/mg dry wt) in the vaginal segment of the cervix was significantly higher than in the mid (P < 0.05) and the uterine (P < 0.01) segments. The hydroxyproline:proline ratio showed the same pattern as the collagen content. The percentage of collagen denaturation in the superficial layer was always significantly (P < 0.01) higher than that in the deep layer, but no effect of the progesterone status or of the segment along the longitudinal axis was seen. It is concluded that regional differences in collagen biochemistry are present in the cervix of nonpregnant cows, which may account for the difference in firmness of different parts along the circular or the longitudinal axis of the cervix. However, differences in texture of the cervix between the two groups of cows could not be explained by differences in the collagen content, percentage of collagen denaturation, or water content.

Immunohistochemical distribution of oestrogen and progesterone receptors and tissue concentrations of oestrogens in the cervix of non-pregnant cows

An immunohistochemical study of the expression of oestrogen (ER) and progesterone receptors (PR) in different regions along the longitudinal and vertical axes of the cervix of non-pregnant cows was performed. Animals were separated into two groups depending on the presence or absence of a functional corpus luteum in their ovaries, as indicated by blood progesterone concentrations. The high progesterone group (HP4) had serum progesterone concentrations > 2.0 ng mL(-1) (n = 6) and the low progesterone group (LP4) had serum progesterone concentrations < or = 0.5 ng mL(-1) (n = 4). Significantly higher concentrations of oestrogen were found in the cervical tissue of animals in the LP4 group than those in the HP4 group (473 +/- 53 v.149 +/- 46 pg g(-1) wet weight; P < 0.01). Furthermore, there was a significant effect of tissue layer (epithelium to deep stroma) on the number of ER (P < 0.01) and PR (P < 0.05) immunoreactive nuclei per 1000 cells. For both ER and PR the proportion of cells expressing the receptor increased from epithelium to subepithelial stroma (P < 0.01) and from subepithelium to deep stroma (ER P < 0.05; PR P =0.061). When the number of receptor-positive cells were expressed per mm2 tissue, differences between the subepithelial stroma and the deep stroma became even more marked. In addition, the vaginal part of the cervix had significantly more (P < 0.01) ER and PR immunoreactive nuclei per 1000 cells than the uterine part, but these differences were no longer apparent when a correction was made for cell density. There was no relationship between progesterone status of the animals, nor local tissue oestrogen concentrations and ER or PR immunoreactivity in the cervix of these non-pregnant cows. Instead, a strong relationship between both longitudinal and vertical positioning of tissue in the cervix and expression of both receptor types was shown. In addition, a strong correlation between ER and PR expression in the subepithelial stroma (R = 0.85, P < 0.01) and the deep stroma (R = 0.83 P < 0.01) was evident. In conclusion, these results demonstrate that in studies of steroid hormone receptor expression in the cervix, careful description of sampling site and depth are necessary if the results are to be interpreted meaningfully.

Ultrasonic cervimetry to study the dilatation of the caudal cervix of the cow at parturition.

The objective of this study was to investigate the temporal changes in dilatation of the caudal cervix during induced calvings (n = 5). We used ultrasound cervimetry, allowing the continuous recording of the distance between a transmitting and receiving ultrasound crystal, which were implanted opposite to each other on the caudal rim of the cervix. We started recording between 19 and 21 h after injecting a prostaglandin analogue (PG) on day 272 of gestation. A fluid-filled catheter had been introduced transcervically between the fetal membranes and the uterine wall for measurements of intra-uterine pressure (IUP). While the characteristics of calving varied widely between the five animals, it appeared possible to divide the process of dilatation into four phases. During the latent phase, which lasted until 25-43 h after PG, no net gain in dilatation occurred. We found an acceleration phase (4.3-6.8 h), in which the dilatation rate speeds up (0.49-0.84 cm/h) in three of the cows. During the phase of maximum slope (lasting 0.5-4.8 h), we measured an even higher rate (1.47-8.48 cm/h), decreasing again during the deceleration phase (rate 0.24-2.28 cm/h) in four cows. The quality of the IUP measurements precluded us from continuously investigating the relationship between cervical dilatation and uterine contractions. However, short term simultaneous recordings revealed that the cervical opening changed momentarily in the absence of IUP during the latent phase, while during the phase of maximum slope, temporary changes of dilatation coincided with uterine contractions. We concluded that the method of ultrasound cervimetry used in this study provides a valuable way to study the process of cervical dilatation in parturient cows in vivo.

A monoclonal antibody recognizing a differentiation marker on rat gonocytes

Abstract Monoclonal antibodies (MAb) were raised against a testicular membrane fraction from 18-day post coitum (p.c.) rat testes. One antibody, designated 4B6.3E10 (μ, kappa), was obtained which specifically reacted with gonocytes in the fetal testis. No significant cross-reactivity with other tissues from the 18-day p.c. embryo was found. MAb 4B6.3E10 was reactive with rat gonocytes from 17-day p.c. until the day of birth. Germ cells at later stages of testis development did not show any labelling. The epitope recognized by 4B6.3E10 is a carbohydrate as periodate treatment leads to a loss of reactivity of the antibody. By SDS-PAGE and Western blotting of proteins extracted from a testicular membrane fraction from 18-day p.c. testes, MAb 4B6.3E10 was found to recognize at least 3 protein moieties with apparent molecular weights in the ranges of 80–100, 120, 160–180 kDa (either under reducing- or non-reducing conditions). The results suggest that MAb 4B6.3E10 recognizes a specific differentiation marker for fetal rat gonocytes.

Immunomagnetic Isolation of Fetal Rat Gonocytes

PROBLEM: An efficient method to obtain highly enriched populations of viable gonocytes from rat embryos at Day 18 and Day 20 postcoïtum (pc) is described.