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

ANIMAL REPRODUCTION SCIENCE ELSEVIER

Animal Reproduction Science 44 ( 1996) 183- 193

Isolation of preantral follicles from nondomestic cats -viability and ultrastructural investigations Katarina Jewgenow, Manuela Stolte

*

Institutefor Zoo Biology and Wildlife Research, D-10315 Berlin, Germany

Accepted 7 February 1996

Abstract Large scale isolation of small preantral follicles (40-90 pm) 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. Keywords:

Preantral

follicle; Nondomestic

cat

1. Introduction Assisted reproduction methods for endangered species are mostly directed toward sperm conservation and artificial insemination procedures. The use of in vitro maturation and fertilisation is limited by the low number of oocytes which can be obtained from

’ Corresponding author at: Institute for Zoo Biology and Wildlife Germany. Tel.: ( + 49) 030/5168-O; fax: ( + 49) 5 126 104. 0378-4320/96/$15.00 Copyright PII SO378-4320(96)01549-7

Research,

PF 1103, D-10252

0 1996 Elsevier Science B.V. All rights reserved.

Berlin,

184

K. Jewgenow, M. Stolte/Animal Reproduction Science 44 (1996) 183-193

ovaries of dead animals. Despite the fact that the mammalian ovary contains a huge number of generative cells, only a few can be cultivated to transferable embryos. The development of systems which support in vitro oocyte growth is valuable for the conservation of the female genetic pool. Following gentle isolation from ovaries, oocytes of preantral follicles must grow to mature stage before they can be fertilised. The growth period of most mammalian oocytes (Gosden, 1992; Gougeon, 1993) is expected to be more than 1 month and successful culturing during all stages of follicular growth presents many challenges. Successful culture of preantral follicles would certainly be of great value for studying early follicular development and its regulation. Alternatively, the genetic potential of preantral oocytes could be conserved by freezing for later transplantation of the preantral follicles into ovaries of an other animal, or by microsurgical transfer of germinal vesicles into mature oocytes. These procedures are possible methods to circumvent a long in vitro growth period. In previous studies, we reported that it is possible to isolate a large amount of preantral follicles from domestic cats (Jewgenow and Goritz, 1995). Secondary follicles obtained from cat ovaries are able to develop in vitro to the antral stage (Jewgenow and Pitra, 1993). This study focused on isolation procedures for primordial and primary follicles (40-90 km diameter) of nondomestic felids in comparison to domestic cats. The ultrastructure of follicles before and after isolation is compared to determine whether morphological changes occur, or species specific features are present.

2. Materials and methods 2.1. Isolation of preantral follicles Ovaries were obtained from five tigers, three lions, one puma, one cheetah and one jaguar who died at zoological gardens and from domestic cats (n = 40) ovariectomised at a local animal clinic (Table 1). Ovaries were collected as fresh as possible, but no later than 8 h after death. Upon removal, ovaries were immediately deposited in Hank’s balanced salt solution (HBSS, Sigma Chemie GmbH, Deisenhofen, Germany) with 1% antibiotic-antimycotic solution (Sigma Chemie GmbH) and processed in the following way. After washing three times in HBSS, the ovaries were dissected into halves and the medulla was removed. The cortex (about l-2 mm thick) was then carefully pressed through a cell dissociation sieve (60 mesh, Sigma Chemie GmbH). This cell suspension was passed through a series of nylon sieves (200, 100 and 40 km). The 40 p,rn sieve was rinsed with 10 ml HBSS to recover all cells with diameters between 40 and 100 p,m. The follicle suspension obtained from some ovaries was centrifuged (300 X g, 5 min) and resuspended in 1 ml culture medium (see below). 2.2. Viability estimation Aliquots of 20 p,l were used to count the number of preantral follicles and to evaluate viability by trypan blue staining (0.05%, 2 min, SERVA, Heidelberg, Germany) of

3 years 16 years

14 months 4 years

10 years

16 years

3- 10 years

Puma Bengal tiger

Siberian tiger Siberian tiger

Sumatran tiger

Sumatran tiger

Indian lion n=3 nondomestic cat n = 11

Asterisks

I-4 years

indicate nonsignificant

domestic cat n=40

I8 years

Jaguar

differences

I .2- I8 years

Poor/ pancreatic disease Poor/cachexia

< 10 years

Cheetah

felids

0.148*0.06 0.139 f 0.04

2.44-6.66

20.93/3.92

4.53/3.34

1.39

1.77

0.95/0.97 1I .4/8.39

0.58,‘3.47

0.40/0.63

Weight right/left (a)

ovary

and domestic

between the means (P > 0.05).

Fair Poor/ gastroentetitis Fair Poor/nephropathy, pyometra Good (death by drowning) Poor/hepatitis, pneumonia Fair

Health status/ pathological indings

Age

Species

Table I Isolation of preantral follicles from ovaries of nondomestic

No

17.3 37.7

150 per ovary 600 per ovary 1867f

Polycystic 2-4 corpora lutea

_

250-24650

per ovary

O-12500 per ovary 2892 f 665 *

28.7 f 3.2

28.2 rt 3.9

No

28.6

1440 per ovary

Without

1144 *

7 days No 18.7 _

21 days

7 days

14 days No

1080 per ovary No

No

Without Without

18.7

No

49.6 26.3

I /2 ovary

32.5

Cultivation

12500 per ovary 100 per ovary

800 from

980 per ovary

Viability in % TB-staining

Cyst > 5 cm polycystic Without Polycystic

Polycystic

Macroscopic structures

No. of isolated preantral follicles

186

K. Jewgenow, M. Srolte/Animal Reproduction Science 44 (1996) 183-193

granulosa cells (Jewgenow and Goritz, 1995). The concentration was adjusted to 1000 vital follicles ml- ’ . Approximately 100 follicles were transferred into prepared four-well culture dishes (NUNC, Roskilde, Denmark) containing 400 ~1 Dulbecco’s MEM supplemented with 0.3% bovine serum albumin (SIGMA Chemie GmbH), and 1% antibiotic-antimycotic solution (SIGMA Chemie GmbH). The follicles were cultured at 375°C in 5% CO, for 6 h in the presence of 10 p,l 5-bromo-Y-deoxy-uridine (BrdU) labelling reagent (0.5 ml l- ’ ; BrdU-Kit II; Boehringer Mannheim Biochemica, Germany). BrdU was added to the culture medium to measure DNA synthesis in granulosa cells and oocytes using a modified procedure for immunocytochemical detection, After the coincubation, the follicles were transferred into siliconised glass tubes containing 10 ml washing buffer, centrifuged (300 X g, 5 min) and resuspended in 50 ~1 buffer. All follicles were mounted on an objective slide and air-dried. The cells were then fixed in 70% ethanol (in glycine buffer, 50 mmol 1-l; pH 2.0) for at least 20 min at -20°C. Further steps were performed according to the manufacturer’s instructions. The slides were washed and subsequently covered with anti-BrdU- and anti-mouse-IgG-alkaline phosphatase working solutions (30 min, 37.5”C). The substrate reaction became visible 30 min after the addition of the substrate solution. The slides were then covered with glycerol-gelatin and evaluated with a light microscope at X 100 magnification. 2.3. Transmission electron microscopy For ultrastructural investigation, specimens of ovarian tissue from three tigers, one puma and several cats were taken immediately after death. In addition, some mechanically isolated preantral follicles of these animals were fixed with 3% glutaraldehyde and were placed in low melting agarose (l%, SERVA Feinbiochemica GmbH, Heidelberg, Germany). After washing with phosphate buffer, post-fixation with osmium tetroxide was performed. The samples were dehydrated in increasing concentrations of ethanol and embedded in Epon 812. For light microscopy, semithin sections (1 km) were cut and stained with toluidine blue. The ultrathin sections (70 nm> were stained with uranyl acetate followed by lead citrate and examined with a Zeiss EM 902 A. 2.4. Statistical analysis Mean values were presented as the standard error of the mean (SEMI. Analysis of variance (ANOVA) was used to test for significant differences between means. P < 0.05 was chosen to indicate significance. All statistical procedures were performed with the software program Statistika for Windows (Release 4.5, copyright StatSoft Inc., 1993).

3. Results 3.1. Isolation of preantral follicles

The mechanical dissection of ovaries from nondomestic cats provided a large number of preantral follicles (o-12500 per ovary) which was similar to the number obtained

K. Jewgenow, M. Stolte/Animal Table 2 BrdU labelling after cultivation

Science 44 (1996) 183-193

of preantral follicles of tigers, lions and cats in Dulbecco’s

Species

No. of cultivated follicles

(no. of animals)

(no. of wells)

Siberian tiger (n = I) Indian lion (n = 3) Domestic cat ( n = 14)

Reproduction

BrdU labelling of

236 (3) 243 (3) 1386(14)

Different letters indicate significant

differences

187

MEM for 7 days

No labelling of follicles

Oocytes

Granulosa

(%I

(WI

12.72 + 7.3a 9.99 +6.2a 12.59 t 2.4a

9.23 +2.6a 25.08 k 9.3b 20.19 +2.lb

cells

(%I

78.0 4 + 4.6 a 64.9 6k 15.0 a 67.2 3 f 3.0 a

between mean values (+ SEMI within columns (P < 0.05).

from domestic cat ovaries (250-24650 per ovary). Despite the fact that the domestic cats were considerably younger, no significant difference between the isolation results of domestic (mean number 2892 + 665) and nondomestic cats (1867 _+1144) was found. The wide variation in the follicle number among individual domestic queens was not correlated to age (the queens were not older than 4 years), cycle, breed or to the season. However, nondomestic cats with very low follicle numbers were either older or had general pathological findings (Table 1). The number of recovered follicles from the

Fig. 1. Puma, native primordial follicle (X 21000) (fee). Note the microvilli (mv) of the oolemma.

with basal membrane

(bm) and follicular

epithelial

cells

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Bengal tiger (16 years old), the 4-year-old Siberian tiger and the 16-year-old Sumatran tiger was 150 or less. Light microscopy revealed that the population was composed of 80-90% primordial and primary (40-50 p_m) follicles with one layer of cells, and lo-20% (60-100 pm> secondary follicles with stratified granulosa cell layers. 3.2. Viability estimation Viability estimated by trypan blue staining after mechanical isolation was 20-50% (Table 1). The number of preantral follicles isolated from the young puma was very high and after 14 days culture, 62.5% follicles were still intact. After 2 weeks of culture, a monolayer of confluent follicle cells was present. The follicles from one Siberian tiger and three Indian lions were cultivated and in vitro viability was assessed by BrdU labelling (Table 2). The percentage of germinal vesicles labelled was 12.7 + 7.7 in the Siberian tiger, 10.0 f 6.2 in the Indian lions and 12.6 + 2.4 in the domestic cats. The percentage of labelled granulosa cells was 9.2 + 2.6 in the Siberian tiger, 25.1 f 9.3 in the Indian lions and 20.1 + 2.1 in the domestic cats. Compared with the Indian lions and the domestic cats, the follicles of the Siberian tiger showed a significant decreased labelling of granulosa cells (9.2%) and lower viability (18.7%) at the beginning of culture (Table 1).

Fig. 2. Puma, isolated primordial follicle (X 21000) with follicular epithelial cells (fee) connected by

desmosomes (d). Ooplasm with mitochondria (m) and vesicles (v).

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Fig. 3. Tiger, isolated primary follicle (X.5GOO)with intact basal membrane (bm) and enlarged intercellular spaces (arrow) between the folliculx epithelium cells. Zona pellucida (zp), ooplasm cop).

3.3. Ultrastructural

comparison

of native and isolated follicles

The isolated primordial follicles appeared like the native ones; consisting of a centrally located oocyte, a single layer of flat follicular cells and a complete basal membrane (Fig. I). The oolemma was smooth and in close contact with the follicular epithelial cells which were joined by desmosomes (Fig. 2). The nuclei of the oocytes were large, round and rich in euchromatin. The nucleoli had a reticular to dense structure. The ooplasm contained mitochondria, vesicles of different size and some ribosomes. At this stage, the endoplasmic reticulum was not well developed and Golgi complexes were rare. In the primary follicles, the oocytes were enclosed by one layer of cuboidal follicular cells and an intact basal membrane. The intercellular spaces within the follicular epithelium of the isolated primary follicles (Fig. 3) were larger than those observed in the native ones. The zona pellucida, up to 10 p,rn thick, was already present at this stage. Cytoplasmic protrusions of the follicular cells and of the oocytes, as well as the microvilli of the oolemma were embedded within it (Fig. 4). In the peripheral region of the ooplasm, Golgi complexes and immature to mature cortical granules were present. Concentric areas of endoplasmic reticulum were developed and free ribosomes, mitochondria and vesicles of different size were randomly distributed. The secondary follicles were characterised by a multilayered follicular epithelium and a thick zona pellucida. At this stage of maturation, no considerable ultrastructural differences between native and isolated follicles were seen.

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Fig. 4. Tiger, isolated primary follicle (X 12000) with zona pellucida (zp) and a cytoplasmic protrusion (arrow). Note the close contact to the oolemma and the central vesicular body (arrow head) in the ooplasm (OP).

Central vesicular bodies were found in the ooplasm of all preantral follicles examined from tigers. They were membrane bound, up to 4 p.m in diameter and contained several granular inclusions of variable size (Fig. 4).

4. Discussion As in domestic cats (Jewgenow and Goritz, 19951, mechanical dissection of ovaries from nondomestic cats provides a large amount of preantral follicles. Comparison of isolation results in felids with those from other species are difficult to perform due to species differences in ovarian composition, variation in follicle classification, and types of follicles included in the final count. However, a greater number of follicles are typically isolated from feline species than from other mammals. For example, in vitro growth of follicles has been most extensively studied in mice and the recovery rate is about 100 follicles per ovary (Wang et al., 1991). Using collagenase for digesting rabbit ovaries, Nicosia et al. (1975) reported the recovery of only 40-60 small primary and 80-100 large primary and secondary follicles. A method of isolating an adequate number of hamster preantral follicles (n = 500) was reported by Roy and Greenwald (1985), and the procedure was later modified to isolate follicles from adult pig (Greenwald and Moor, 1989) which resulted in recovery of up to 30000 primordial follicles per ovary. Seventy-five percent of these follicles were naked and smaller than

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30 km. The number of preantral follicles isolated from bovine ovaries was about 3000 per foetal ovary (Figueiredo et al., 1993; Sirard and Coenen, 1995) and 30-500 per calf or cow ovaries (Jewgenow and Pitra, 1991; Nuttingk et al., 1993). Germ cells from nondomestic animals are only available from females which die or are slaughtered, or ovariectomised for medical reasons. Therefore, most data were often from older and/or medically compromised individuals. The number of isolated follicles varied markedly within and among species, but no correlation to age and/or health status was found. The recovery of follicles from the young puma (3 years old) was superior to most domestic cats (mean 2892) post-isolation of the preantral follicles was highly viable and after 14 days culture, 62.5% follicles were still intact. In contrast, the LCyear-old Siberian tiger was the only animal from which follicles were not recovered. It was possible to collect oocytes from older females including those with pathological changes of the ovaries. Nondomestic cats, older than 12 years can probably be considered reproductively senescent (Johnston et al., 199 I ). Polycystic ovaries were found in four individuals aged lo-18 years; the recovery rate from these ovaries (100-1600) and post-isolation viability was comparable to that from the other individuals. The ‘domestic cat model’ is suitable for developing new techniques of assisted reproduction which can be applied to rarer species (Wild& 1991; Pope et al., 1993). Ovaries from ovariectomised cats are available from veterinary clinics and fundamental research on germ cell biology can be conducted without ethical barriers. It appears that genera1 in vitro techniques developed in one felid species are largely adaptable to another species. However, certain species specific characteristics are to be expected (Wildt, 1991). For example, sperm viability of the snow leopard is highly sensitive to the complex media, which is used for sperm treatment in cats (Roth et al., 1994). This was the first comparative transmission electron microscopic study of feline preantral follicles. Our results demonstrate that no fundamental species specific differences exist between the preantral follicle ultrastructure of cats, tigers and pumas. The morphology of feline oocytes appears similar to that of most mammalian species (Wassermann, 1988; Leiser, 1990; Fawcett, 1994). However, a central vesicular body was observed in tiger oocytes. The function and possible species specific role is unknown. The mechanical isolation procedure resulted in 50-80% loss of follicle viability as estimated with trypan blue staining. Even follicles recovered from domestic cats, whose ovaries are considerably smaller than ovaries from all other felids, are characterised by only 28.7% intact follicles. However, due to the tight contact between the oocyte and surrounding granulosa cells (Figs. 2 and 4), staining of the entire follicle after injuring only some of the cells is expected for a short period. BrdU labelling of cells appears to be a better method to evaluate viability during isolation and short term cultivation. In domestic cat, 12.6% of the cultured oocytes and 20.2% of granulosa cells were labelled with BrdU. The surprising labelling of approximately one-third of the oocytes can be explained by extensive mitochondrial growth and division throughout growth, which is certainly accompanied by mitochondrial DNA synthesis. The preantral follicles from the nondomestic cats showed a similar labelling rate of oocytes. However, the granulosa cells from one tiger were less proliferative (9.23% granulosa cells with BrdU incorpora-

192

K. Jewgenow, M. Stolte/Animal Reproduction Science 44 (1996) 183-193

tion). However, not enough data from other individuals and species were available to correlate these findings. Our transmission electron microscopic study revealed that the mechanical isolation had no impact on the morphology of preantral follicles. No considerable ultrastructural changes were observed in primordial, primary and secondary follicles after the mechanical dissection of ovaries and the isolation procedure. The native, as well as the isolated preantral follicles, were surrounded by a complete intact basal lamina. Slight morphological differences, such as the enlarged intercellular spaces of the follicular epithelium seen in the isolated follicles, may be caused by the processing. In conclusion, the ability to preserve female germ cells from rare animals is an important prerequisite for introduction of modem reproductive techniques into conservation programs. The recovery of considerable amounts of viable preantral follicles from ovaries of nondomestic felids is only the first step. Further fundamental research concerning cryopreservation and in vitro growth is necessary. The use of domestic cat as an experimental model to study methods for artificial assistance and control of reproduction in nondomestic feline species seems to be a worthy approach.

Acknowledgements We are grateful for the valuable help of the pathologists and especially to Hen-n Bemd Paschmionka for taking the samples. We also thank Dagmar Viertel, Marion Biering as well as Doris Fichte for technical assistance and Dr. Chris Talsness for correcting the English manuscript.

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