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Ovarian follicles are a major site of oestrogen synthesis in sheep, but almost nothing is known about the relative contribution made by follicles of different sizes to the output of oestrogen at various stages of the reproductive cycle. To determine this, ovaries were dissected from sheep, all follicles over 2 mm in diameter were explanted separately as organ cultures and the daily production of oestrogen from each follicle was measured. This study was preceded by a series of preliminary experiments undertaken to define suitable conditions in which ovarian follicles would retain both their morphological integrity and steroidogenic capacity in culture.
The 224 follicles used in the preliminary series of experiments were explanted from the ovaries of seventeen sheep killed between the 2nd and 15th days of the oestrous cycle. A
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Summary.
A total of 429 eggs was used to investigate procedures both for the removal of the zona pellucida and for the culture of sheep eggs in vitro.
The effect of pronase, papain, bromelain, trypsin and α-chymotrypsin, on the zonae pellucidae of fertilized and unfertilized eggs was studied in the first series of experiments. All the enzymes were effective in removing the zonae pellucidae from unfertilized eggs. Pronase was the only enzyme that had a significant lytic effect on the zonae of fertilized eggs denuding 34% of eggs in less than 5 min and almost all the other eggs within 90 min.
Investigation of some of the factors involved in the successful culture of normal sheep eggs in vitro constituted the second part of the study. Eggs of both the eight- to sixteen-cell and morula stages were cultured for 72 hr in either sheep serum, enriched tissue culture medium 199 or Brinster's medium. The development and viability of the eggs after culture was determined by transferring some eggs to recipient sheep and making accurate cell counts on the remainder. Eggs recovered at the eight-cell stage underwent only one or two cleavage divisions during the 72-hr culture period. These eggs did not develop into foetuses when transferred to the uteri of recipient sheep. On the other hand, the majority of morulae cultured for 72 hr in enriched M199 or sheep serum underwent cleavage and blastulation in vitro. That the cultured blastocysts were normal was demonstrated by their implantation and growth into foetuses when transferred to the uteri of suitable recipients.
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The gonadotrophin (pmsg) present in the serum of mares between the 40th and 120th days of pregnancy originates in the uterine endometrial cups. These structures begin to develop on the 36th day of pregnancy opposite a transitory, though well-defined, circumferential thickening of the chorion called the allantochorionic girdle (Ewart, 1897; van Niekerk, 1965; Allen, 1970). The endometrial cups are composed of a discrete and densely packed mass of very large, epithelioid, decidual-like cells. They develop before the allantochorion becomes attached to the endometrium and it has been widely accepted in the past that they are exclusively maternal in origin (Clegg, Boda & Cole, 1954; Amoroso, 1955). However, recent genetic evidence, derived from the study of interspecific equine hybrids, has indicated
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Summary. The pattern, chemical nature and biological role of polypeptides secreted by ovine follicles exposed to gonadotrophins in vivo were studied. Follicles were removed at intervals before ovulation (in-vivo groups), separated into granulosa and cumulus compartments, and incubated for 3 h with radiolabelled amino acids. No differences were detected in the polypeptides (M r 46 000–60 000) secreted by granulosa and cumulus cells before exposure to the preovulatory LH surge. Each class of polypeptides was characterized by different degrees of phosphorylation, sulphation and glycosylation. Within 15 h of the LH surge the secretion of the M r 46 000–60 000 polypeptides had ceased and was replaced by a non-sulphated M r 30 000 secretory product. Significant differences in the secretory pattern of granulosa and cumulus cells were detected after exposure to LH.
Intact follicles and granulosa cells were cultured for 24 h (in-vitro groups) and then incubated with radiolabelled amino acids. The profile of polypeptides secreted by intact follicles cultured in the presence or absence of LH corresponded closely with the profile observed in vivo. By contrast, granulosa cells grown as monolayers switched spontaneously to the secretion of M r 30 000 polypeptides in medium devoid of gonadotrophin. This aberrant secretory switch did not occur in granulosa cells maintained in suspension culture. Inhibition of transcription in follicles exposed to LH prevented both the appearance of the M r 30 000 polypeptide and the disappearance of the M r 46 000–60 000 polypeptides. Although the inhibition of steroidogenesis by a variety of steroid enzyme inhibitors was without effect on secretion, evidence was obtained to suggest that one of the secreted polypeptides binds oestradiol-17β
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Summary. The susceptibility of sheep oocytes to temperature changes during maturation in vitro was tested by reducing the incubation temperature to 20°C at various stages of meiosis. Cooling induced chromosomal abnormalities including disorganized metaphase plates and multipolar spindles in 28–54% of oocytes cooled at all stages of meiosis from germinal vesicle breakdown (GVBD) to metaphase II. The time of GVBD (8–11 h after the start of culture) was the most sensitive to cooling, whereas fewest abnormalities were found in oocytes cooled in late metaphase I (16–19 h). In addition to the chromosomal abnormalities, unusual vesicles appeared in the cytoplasm of oocytes cooled at 8–11 h and 12–15 h. No abnormalities in protein synthesis were detected by one-dimensional SDS gel electrophoresis.
The consequences of the abnormalities for the developmental potential of the cooled oocytes were tested by transfer to recipient ewes and fertilization in vivo. After 12 days of development only 6% and 11% oocytes cooled at 12–15 h and 20–23 h respectively had developed to expanded blastocysts, compared with 44% of control oocytes.
The results demonstrated that maturing sheep oocytes are very sensitive to a drop in temperature.
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Summary. Ovine granulosa cells respond, through transcriptional mechanisms, to preovulatory concentrations of gonadotrophins by secreting an M r 30 000 polypeptide. To determine the stages of the oestrous cycle at which this polypeptide is secreted, corpora lutea were collected on Days 3, 7, 10, 13 and 16 (Day 0 = oestrus; n = 4 per group), cut into 1-mm slices, and incubated for 6–7 h with [35S]methionine. Radiolabelled polypeptides of intra- and extra-cellular origin were separated by polyacrylamide gel electrophoresis and quantitated by densitometry. The M r 30 000 polypeptide was secreted at all stages of the luteal phase tested (Days 3–16), and represented approximately 24% of the total labelled polypeptide present in the medium; polypeptides of approximate M r 14 000, 25 000 and 46 000 accounted for most of the other secreted proteins. Neither pituitary hormones (LH, FSH, prolactin) nor cholera toxin (chosen to activate adenylate cyclase) affected the rate of production of M r 30 000 polypeptide, indicating that, once secretion has been initiated in the granulosa cells, it is not readily modulated by hormonal intervention after luteinization.
Incubation of luteinized granulosa cells with tunicamycin (inhibits N-linked glycosylation reactions) showed that the secreted polypeptide consists of a heavily glycosylated amino acid backbone of approximately M r 20 000. Western blot analysis established further that the polypeptide was not an inhibin subunit. However, NH2-terminal amino acid sequencing of the first 25 amino acids revealed a 68% sequence identity between the secreted polypeptide (M r 30 000) and a human tissue inhibitor of metalloproteinases.
Keywords: granulosa cell; corpus luteum; polypeptide secretion; metalloproteinase inhibitor; sheep
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Summary. Oocytes removed from, or retained within, non-atretic and atretic follicles of different sizes were cultured for 24 h in the presence of a variety of hormones in an attempt to identify the factors affecting oocyte maturation in vitro. Resumption of meiosis was assessed morphologically; the developmental capacity of oocytes after culture was determined by transfer to the oviducts of inseminated ewes.
About 70% of oocytes cultured after removal from follicles of different sizes resumed meiosis in vitro, but they did not undergo normal development after transplantation.
Oocytes cultured within the follicle in hormone-free medium remained at the germinal vesicle stage. In the presence of FSH and LH some oocytes reached the second meiotic metaphase: 19% in small (2–3 mm diam.) and 73% in larger (3–5 mm diam.) non-atretic follicles, and 54% in small and 45% in larger atretic follicles.
Less than 5% of oocytes cultured in follicles developed into normal blastocysts after transplantation when either no hormone or only FSH and LH were added to the culture medium. The addition of oestradiol-17β to medium containing FSH (2 μg/ml) and LH (1 μg/ml) resulted in the development to blastocysts of 26% of oocytes from small non-atretic follicles, 46% from large non-atretic follicles and 50% from atretic follicles. Blastocyst formation was greatly depressed and fragmentation rate significantly increased with concentrations of 10 μg FSH/ml and 2 μg LH/ml.
Developmental capacity after culture was further demonstrated by the birth of lambs from 63% of blastocysts derived from oocytes matured in vitro; 52% of control blastocysts developed to lambs after transfer.
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It has been well established that removal of the uterus in many mammalian species results in the prolongation of the function and life-span of corpora lutea (Bland & Donovan, 1966; Anderson, Bland & Melampy, 1969; Caldwell, Rowson, Moor & Hay, 1969; Schomberg, 1969; Caldwell, 1970). Furthermore, most evidence suggests that uterine transplantation to ectopic sites can at least partially reverse the extended life-span of the corpora lutea in hysterectomized animals (see Caldwell, 1970, for review). When these studies were extended to test various extracts of uterine tissue or flushings, however, the results were not conclusive. Both success and failure have been reported in the attempt to influence corpus luteum function in hysterectomized animals using extracts of uterine origin given by several routes of administration in various test animals and bioassay systems (Schomberg, 1969; Caldwell, 1970). Previous work has also
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Summary. To examine the effects of somatic cell support on the cleavage and viability of fertilized sheep eggs, 434 pronucleate eggs were co-cultured for 3 or 6 days on oviduct cells or fibroblasts and 77 eggs were cultured in medium alone. During the first 3 days in culture 95% of the single-celled eggs cleaved regularly to non-compacted morulae on either of the feeder-layers but only 13% underwent similar regular cleavage in medium alone. Despite the identical cleavage rates in the co-culture groups, only 33% of embryos grown on fibroblasts as compared with 80% of embryos grown on oviduct cells were fully viable as judged by their ability to develop normally after transfer to recipient animals. The viability of embryos in the oviduct group was equal to that obtained after the direct transfer of morulae from donor to recipient sheep.
After 6 days in culture 42% of embryos co-cultured with oviduct cells developed into expanded blastocysts as compared with only 4·5% cultured on fibroblasts. In both co-culture groups virtually all the remaining embryos blocked during the 4th cleavage. When transferred, 30% of blastocysts grown from the pronucleate stage on oviduct cells were viable.
We conclude that: (1) during the first 3 days after fertilization cleavage will progress at a normal rate on different feeder-layers but oviduct cells appear to be required for the acquisition of full embryonic viability; (2) in our system oviduct cells are able to support passage of embryos through the critical 4th cell cycle, whilst fibroblasts are almost entirely unable to support this critical phase; and (3) it is apparent that the factors necessary for the morphological formation of the blastocyst may be insufficient or different from those which endow it with subsequent developmental ability.
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Summary. An enzymic method for recovering primordial follicles from the pig ovary consists of incubating cortical slices for 2 h with 0·025% collagenase 1A. An average of 185 000 or 419 000 primordial follicles per ovary were recovered from ovaries collected in Cambridge and Kansas, respectively. Following a discontinuous Percoll gradient, primordial follicles can be separated from contaminating somatic cells by mouth pipette or a micromanipulator to collect 100–1500 follicles but for large scale recovery of approximately 30 000 follicles flow cytometry is recommended. Two types of primordial follicles can be distinguished by electron microscopy: peripheral clusters of small oocytes with an incomplete investment of pregranulosa cells and a deeper region of individual oocytes surrounded by a complete layer of pregranulosa cells. The viability of the purified primordial follicles is attested by their ability to synthesize proteins for at least 12 h after incubation with [35S]methionine. Moreover, the primordial follicles showed several polypeptide bands in common with mature oocytes especially with M r of 60 000–90 000 but with considerable differences from somatic cells.
Keywords: primordial follicles; structure; protein synthesis; enzymic dissociation; pig
