Changes in water content, collagen degradation, collagen content, and concentration in repeated biopsies of the cervix of pregnant cows.

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 alpha-chymotrypsine-treated samples. By incorporating a hydroxyproline standard series in the measurements, the insoluble collagen content (mug/mg dry wt) as well as the insoluble collagen concentration (mug/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.

Regional differences in water content, collagen content, and collagen degradation in the cervix of nonpregnant cows.

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 alpha-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 (mug/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.

Expression of the scaffolding subunit A of protein phosphatase 2A during rat testicular development.

Previously, we found that the poly(A)+ RNA of the scaffolding subunit A (alpha isoform) of protein phosphatase 2A (PP2A-Aalpha) was clearly expressed by fetal gonocytes but weakly expressed by adult single (As), paired (Apr), and aligned (Aal) A spermatogonia. The scaffolding subunit A of PP2A (PP2A-A) is the major subunit in the formation of a functional PP2A holoenzyme. In this study, we investigated the expression of PP2A-A during testicular development in more detail using in situ hybridization, immunohistochemistry, and Western blot with testes of rats of various ages from 16 days postcoitum (pc) to adulthood. The expression of PP2A-A was detected in fetal proliferative gonocytes at 16 days pc, declining thereafter during the quiescent period of the gonocytes. From the day of birth to the start of spermatogenesis (Day 4 postpartum [pp]), the number of PP2A-A-immunopositive gonocytes increased. At Day 4 pp, the first A1 spermatogonia appeared along the basement membrane; all were PP2A-A positive. In the adult, PP2A-A was upregulated during the differentiation of the As, Apr, and Aal spermatogonia to the A1 spermatogonia and expressed thereafter by all other spermatogonia. Spermatocytes from the pachytene stage onward and all spermatids in the adult testis also showed clear expression of PP2A-A. In Sertoli cells, PP2A-A was detected during their proliferative period at 19 days pc to 15 days pp. The presence of a functional enzyme was confirmed by the additional detection of the catalytic subunit C of PP2A using Western blot analyses at various ages during testicular development. This apparent pattern of expression of PP2A-A during testicular development suggests that PP2A may play an important role in the proliferation of distinct populations of testicular cells and during meiosis and sperm maturation.

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.

Purified gonocytes from the neonatal rat form foci of proliferating germ cells in vitro.

Due to the lack of a specific marker for gonocytes from newborn rats, isolation of these cells has proven difficult and laborious. We have found a specific cell membrane marker, the Epithelial Cellular Adhesion Molecular (Ep-CAM) that can be used to isolate these cells using antibody directed cell sorting. 4 days post partum (dpp) rat testes were enzyme treated to attain a cell suspension, which was labelled with an antibody (GZ1) against Ep-CAM and tagged with a fluorescent probe. The labelled cell suspension was run through a FACS cell sorter, from which a gonocyte suspension of >85% purity was attained. The cells remained viable in culture and proliferated actively as determined by double labelling the cells with anti-HSP90alpha (a specific germ cell marker) and anti-BrdU antibodies (after BrdU incorporation). During culture, these cells formed chains of 2 to 4 cells and aggregates of proliferating germ cells were found after 8 days of culture.

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.

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.

Spermatogonial stem cell transplantation.

The development of the spermatogonial transplantation technique has given new impetus to research on spermatogonial stem cells. Possibilities opened by this technique include: (a) New ways to study fundamental aspects of spermatogenesis; (b) Generation of transgenic large domestic animals; (c) Protection of (young) male cancer patients from infertility due to chemotherapy or radiotherapy. Spermatogonial stem cell transplantation for the above purposes encompasses a number of steps. First, the stem cells have to be isolated and possibly purified. Second, it should be possible to cryopreserve the stem cells, for example till the children have reached puberty. Third. it should be possible to culture spermatogonial stem cells for a prolonged period of time which would also allow transfection and subsequent selection of stably transfected cells. Fourth, in case of animal studies. the host testis should be emptied from endogenous stem cells. This is probably best done by local irradiation. Finally, the stem cells will have to be transplanted.

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 postcoitum (pc) is described. METHOD: Single-cell suspensions with high cell yield were obtained by a collagenase/ trypsin digestion of the decapsulated testis. The gonocytes were purified by a direct immunoseparation technique, using magnetizable beads coated with rat anti-mouse immunoglobulin M (IgM) and a monoclonal antibody 4B6.3E10, which specifically reacted with a differentiation antigen on the fetal germ cells. RESULTS: Populations of 8.3 +/- 2.7 (x10(3); 18 days pc) or 1.2 +/- 0.25 (x10(4); 20 days pc) viable gonocytes per testis with purities of 91 +/- 6.5% and 92 +/- 4.3%, respectively, as determined by Nomarski microscopy were obtained. CONCLUSION: The cells were successfully used for culture studies and as starting material for the investigation of gene expression.

Ontogeny and localization of an oncostatin M-like protein in the rat testis: its possible role at the start of spermatogenesis.

Oncostatin M (OSM), a member of the interleukin 6 family of cytokines, was found to be highly expressed in the late fetal and early neonatal rat testis, as well as in the maturing and adult testis. Two different forms of OSM were observed, one of M(r) 22,000 and the other of M(r) 36,000. In the prepubertal rat testis [19 days post coitum, 8 days post partum (dpp), and 15 dpp], the form with the higher molecular weight prevailed, whereas in the maturing testis (30 dpp, 45 dpp, and 12 weeks post partum), a shift toward the lower molecular weight form was observed, as well as a decrease in its relative amount. By immunohistochemistry on testicular sections, OSM-specific immunostaining was observed in the interstitial tissue at every age studied. In contrast, OSM immunoreaction was localized in the Sertoli cells exclusively around the start of spermatogenesis, being strongest at 3 dpp. In vitro studies revealed that neonatal Sertoli cells produce OSM. The possible role of OSM at the start of spermatogenesis was investigated by using a coculture of Sertoli cells and gonocytes isolated from newborn rats. OSM significantly increased the survival of both Sertoli cells and gonocytes in a dose-dependent manner. The proliferative activity of the Sertoli cells was not affected by OSM, whereas that of gonocytes was increased by almost 60% after 6 days of culture. Comparison of the effect of OSM on these cocultures with other members of the interleukin 6 family of cytokines demonstrated that this factor is more potent than leukemia inhibitory factor or ciliary neurotrophic factor. On the basis of these findings, it can be concluded that OSM is present in the rat testis, and it is likely to play an important role at the start of spermatogenesis.

Isolation of the synchronized A spermatogonia from adult vitamin A-deficient rat testes.

A method for isolating A spermatogonia from the adult vitamin A-deficient (VAD) rat testis is described. After removal, the testes were decapsulated and tubules were dissected. An enzymatic digestion with collagenase, hyaluronidase, and trypsin was performed first to eliminate most of the interstitial cells. A second digestion with collagenase and hyaluronidase was performed to obtain a cell suspension with a high number of A spermatogonia. The cell suspension was further enriched with A spermatogonia by preplating on peanut agglutinin and separating on a discontinuous Percoll gradient. By this procedure, purification of the suspension to 70-90% A spermatogonia was obtained. In the seminiferous tubules of the VAD rats, only Sertoli cells, A spermatogonia, and some preleptotene spermatocytes are present. In our rats, the A spermatogonia are almost all arrested in the G1 phase of the cell cycle before the S phase of A1 spermatogonia, and presumably before their differentiation into A1 spermatogonia. After administration of vitamin A, spermatogenesis starts synchronously from these A spermatogonia. The isolation of these synchronized A spermatogonia opens ways to investigate the regulation of differentiation and proliferation of A spermatogonia and the biochemical characteristics of the subsequent types of A spermatogonia.

Leukemia inhibitory factor and ciliary neurotropic factor promote the survival of Sertoli cells and gonocytes in coculture system.

Leukemia inhibitory factor (LIF) and ciliary neurotropic factor (CNTF) were found to be pleiotropic modulators of Sertoli cell and gonocyte development (both isolated from the neonatal rat testis) in a coculture system, whereas IL-6, another member of this cytokine family, had no effect on these cells. LIF and CNTF significantly enhanced the survival of the Sertoli cells in a dose- and time-dependent manner. The effect of LIF on the Sertoli cells was significant at a concentration of 1 ng/ml after 3 or 6 days of culture, whereas CNTF had a significant effect at 10 ng/ml. Neither LIF nor CNTF had an effect on Sertoli cell proliferation. The survival of proliferating gonocytes (isolated from 3-day-old rats testes) was also significantly higher in cultures to which LIF (7.5 ng/ml) or CNTF (10 ng/ml) was added. No effect of these cytokines was found on the mitotic activity of proliferating gonocytes. However, LIF (7.5 ng/ml) stimulated the proliferation of quiescent gonocytes (isolated from day 1 testes) after 3 days of culture. Combinations of LIF (or CNTF) with fibroblast growth factor 2 (10 ng/ml) and steel factor (50 ng/ml) did not further improve the long term culture of the gonocytes. LIf- and CNTF-like proteins of the expected molecular masses (32,000 and 22,000 daltons, respectively, under reducing conditions) were found by Western blotting in testicular extracts of 3-day-old rats. Taken together, these results indicate that LIF or CNTF may play a role at the start of the spermatogenesis. The characterization of receptors for LIF or CNTF on the gonocytes and/or neonatal Sertoli cells will aid in a better understanding of the physiological role of these cytokines in the reproductive system.

Effect of fibroblast growth factor-2 on Sertoli cells and gonocytes in coculture during the perinatal period.

Sertoli cell-gonocyte cocultures obtained from rat testes 20 days postcoitum, 1 day postpartum, and 3 days postpartum were used to investigate the effect of FGF-2 on both somatic and germ cells in vitro during the perinatal period. With cells isolated from fetal, newborn, or 3-day-old animals, FGF-2 was found to significantly increase the number of Sertoli cells after 3 or 6 days of cultures, starting at a concentration of 1 ng/ml. FGF-2 did not increase the [3H]thymidine labeling index of Sertoli cells, indicating that FGF-2 is a survival factor for these cells in vitro. FGF-2 (1, 5, or 10 ng/ml) also significantly increased the number of gonocytes after 6 days of culture with cells from either newborn or 3-day-old animals. About twice as many germ cells were found in those cultures compared to the control cultures. Addition of a neutralizing antibody against FGF-2 to control cultures caused a significant decrease in the number of gonocytes compared to that in untreated cultures after 6 days, whereas with FGF-2, the antibody decreased the number of germ cells to control levels. FGF-2 significantly stimulated the proliferative activity of the gonocytes after 3 or 5 days, indicating that FGF-2 is a survival as well as a mitogenic factor for these cells. Taken together, these data suggest that FGF-2 is an important factor around the start of spermatogenesis, at least in vitro.

Characteristics of A spermatogonia and preleptotene spermatocytes in the vitamin A-deficient rat testis.

The proliferative activity and other characteristics of germ cells in the vitamin A-deficient (VAD) rat testis were investigated. In the VAD testis, A spermatogonia and preleptotene spermatocytes were found. The A spermatogonia in the VAD testis showed a bromodeoxyuridine (BrdU) labeling index of 6.6 +/- 1.1% and a mitotic index of 2.8 +/- 0.5%. After continuous labeling with BrdU for up to four days, the ultimate labeling index of A spermatogonia was 11.6 +/- 2.5%, which is less than expected. It is concluded that in the VAD rat testis, many of the proliferating A spermatogonia degenerate. During the first 18 h after administration of vitamin A, no increase was observed in either the labeling index or the mitotic index of the A spermatogonia. However, after 24 h the first wave of A spermatogonia in S phase was found, and the first wave in mitosis was found after 48 h. Furthermore, in the VAD testis the DNA content of most of the A spermatogonia was similar to that of Sertoli cells, i.e., 2n. It is concluded that in the VAD situation, nearly all A spermatogonia are arrested before the S phase of the A1 spermatogonia. The hypothesis is put forward that in the VAD testis, the remaining A spermatogonia are the undifferentiated spermatogonia that are unable to differentiate into A1 spermatogonia. The preleptotene spermatocytes in the VAD testis showed a BrdU labeling index of 20.3 +/- 3.5%, while the DNA content of most of these cells was between 3n and 4n.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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 cycle and for stimulating the formation of A spermatogonia, a process characterizing the start of spermatogenesis.

A monoclonal antibody recognizing a differentiation marker on rat gonocytes.

Monoclonal antibodies (MAb) were raised against a testicular membrane fraction from 18-day post coitum (p.c.) rat testes. One antibody, designated 4B6.3E10 (mu, 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.

Significant improvement of the survival of seminoma cells in vitro by use of a rat Sertoli cell feeder layer and serum-free medium.

Seminoma cell lines, essential to the study of the biology of seminoma, do not exist. Tissue culture conditions for establishing such cell lines have to be developed. Under conventional culture conditions, seminoma cells usually die within the first 3 days after plating. The enhanced survival of rat gonocytes when cocultured with rat Sertoli cells in serum-free medium suggests that seminoma cells, the neoplastic counterparts of gonocytes, might benefit from the same conditions. Indeed, when cocultured with rat Sertoli cells in a serum-free medium, viable seminoma cells could be demonstrated on the 11th day of culture. This result is a significant improvement over the results with conventional methods.

Inhibin reduces spermatogonial numbers in testes of adult mice and Chinese hamsters.

Bovine follicular fluid (bFF) injected ip in mice during 2 days (65,000 U inhibin/day, 1 U inhibin the activity in 1 micrograms bFF protein) caused a significant decrease in the numbers of A4, intermediate (In), and B spermatogonia to 91%, 74%, and 67% of the control values, respectively. The numbers of undifferentiated spermatogonia remained unchanged. These injections suppressed peripheral FSH levels to 6% of the control values, suggesting that FSH might be the modulator of the effects on spermatogenesis. However, in the Chinese hamster, intratesticular injections of bFF during 4 days (6500 U inhibin/day into one testis) also caused a significant decrease in the numbers of A3. In, B1, and B2 spermatogonia to 86%, 61%, 55%, and 94% of the control values, respectively. Similarly, treatment with a partially purified inhibin preparation from rat Sertoli cell-conditioned medium (rSCCM) during 4 days (Mono Q fraction; 1512 U inhibin/day; 37.8 micrograms protein) caused a significant decrease in the numbers of A3, In, B1, and B2 spermatogonia to 90%, 87%, 66%, and 93% of the control values, respectively. Treatment with a highly purified inhibin preparation from rSCCM during 4 days (30K inhibin; 750 U inhibin/day; 100 ng protein) significantly decreased the numbers of In and B1 spermatogonia to, respectively, 87% and 91% of the control values. These effects were limited to the testis into which the material was injected; the contralateral testis or testes injected with control fluid always showed normal numbers of spermatogonia. This implies that the effects on the seminiferous epithelium are not FSH mediated. Intratesticular injections of bFF or pure inhibin did not affect the number of undifferentiated spermatogonia. However, the Mono Q fraction caused a significant increase in the numbers of undifferentiated spermatogonia in stages IV-VII of the cycle, suggesting the presence of a mitogenic factor for undifferentiated spermatogonia in rSCCM which is not present or is counteracted in bFF. The results suggest that inhibin may have a role in the regulation of spermatogonial development in the adult animal.

Isolation of rat gonocytes by velocity sedimentation at unit gravity.

A method for obtaining enriched populations of gonocytes from rat embryos at 18 days p.c. has been developed. Single cell suspensions with high cell yield and good viability of the cells were obtained by a collagenase/trypsin digestion of the testes. Cells were separated on the basis of size by the Staput technique of velocity sedimentation at unit gravity. Populations of 600,000 gonocytes (70-75% purity), sedimenting at about 12 mm/h, could be obtained from 30-35 fetal rats within 8 h after killing. Purities were determined by Nomarski microscopy and verified in fixed preparations and by Coulter volume measurements.