Z. Zellforsch. 97, 30--37 (1969)

Continuities Between Mitochondria and Endoplasmic Reticulum in the Mammalian Ovary

JOHN R. RUBY, ROBERT F. DYER, and RICHARD G. SKALKO

Department of Anatomy, Louisiana State University School of Medicine New Orleans, Louisiana 70112

Received January 20, 1969

Summary. An electron microscope study of developing mouse oocytes has revealed a close morphological relationship between mitochondria and endoplasmic reticulum. In many instances, it was noted that the outer mitochondrial membrane was continuous with the reticular membranes. These cytoplasmic membranes are smooth or studded with ribosomes. These continuities establish an open channel between the endoplasmic reticulum and mitochon- dria. Similar connections are also found in isolated preparations of mitochondria from the adult guinea pig ovary. The functional significance of these observations are discussed in relation to biochemical studies which demonstrate a transfer of protein from endoplasmic reticulum to mitochondria.

A close relationship of endoplasmic re t iculum to mitochondria has been noted in the developing oocytes of hamster (WEAKLEY, 1967), guinea pig (ADAMS and HERTIG, I964), r abb i t (ZAMBONI and MASTROIANNI, 1966), and h u m a n (HERTIG and ADAMS, 1967) and in the hiteal cells of the ra t (CoH]~R]~ et al., 1967), bu t no significance has been placed upon this observation. The demonst ra t ion of a direct connect ion (RoBERTSON, 1961) and a fusion (ANDRe, 1962) of ret icular membranes with the outer mitochondrial membrane has led other invest igators to speculate t ha t the cytoplasmic membrane system contr ibutes to the format ion of mitochon- dria. As suggested by BOWMA~ (1967), the direct con t inu i ty of these membranes may provide a pa thway for the t ransfer of mater ial to or from the mitochondria . This la t ter viewpoint has been substant ia ted , in part , by an autoradiographic s tudy (DRoz, 1967) and by several biochemical invest igat ions (KADENBACH, 1966; HAZDAR et al., 1967). The biochemical studies have shown tha t m a n y of the mitochondrial enzymes are first synthesized on the microsomes and subsequent ly transferred to mitochondria. The present report also lends support to this hypo- thesis by demonst ra t ing the morphological channel through which such a t ransfer may he accomplished.

Materials and Methods

The animals used in the in situ study were ICR albino mice. Ovaries were removed from the animals at 17 and 18 days after fertilization and at 24-hour intervals during the first week of postnatal life beginning on the day of birth. A total of 22 litters were processed. The ovaries were fixed in toto in either: 1.1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.3) or 2. 4% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) followed by numerous rinses in 0.1 M phosphate buffer containing 0.2 M sucrose. All material fixed in glutaraldehyde was post-osmicated. Following fixation, the ovaries were passed through a graded series of alcohols and embedded in Maraglas (FREEMAN and SrUI~LOCK, 1962). The tissue was sectioned on an LKB ultratome or a Reichert OM-U2 ultramicrotome. The sections were mounted on uncoated grids and stained with uranyl acetate (WATsO~r 1958) and lead citrate (REYNOLDS, 1963). All specimens were viewed with a Philips 200 electron microscope.

Mitochondria were isolated from ovarian tissue by the following procedure. Ovaries from six adult guinea pigs were pooled and homogenized in 0.88 M sucrose. The homogenate was centrifuged at 5,000 rpm in a Spinco Model L Ultracentrifuge (No. 40 rotor). The resulting

Mitochondrial - - ER Continuities 31

Fig. 1. Electron micrograph showing a cluster of mitochondria in a mouse oocyte. Note mitochondria with no cristae (NC), with typical cristae (TC), and with intracristal vacuoles (IV). Profiles of endoplasmie reticulum may be associated with more than one mitoehon- drion (A), and a mitochondrion may have a close relationship to more than one segment of the reticulum (B). Mitochondria are also in apposition to short profiles of the reticulum (C) and with vacuoles (D). x 25,000. All tissues in these micrographs were fixed in glutaraldehyde

with postosmication

32 J .R. RvBY, R. F. DYER, and R. G. SKALKO : Mitochondrial - - ER Continuities

pellet was discarded and the supernatant was centrifuged at 12,500rpm (14,000• for 15 minutes. This pellet was resuspended in 0.88 M sucrose and centrifuged at 12,500 rpm. The pellet of mitochondria was processed for electron microscopy as described above.

Observations

The salient ultrastructural features of mouse oocytes do not differ significantly from other mammalian oocytes which have been described previously by other investigators (WEAKLEY, 1967; ADAMS and HERTIG, 1964; ZAMBONI and MAS- TROIANN~, 1966; HERT~G and ADAMS, 1967). These oocytes contain a rather large volume of cytoplasm but possess a relative sparcity of cytoplasmic organelles. I t was not difficult, therefore, to note that almost without exception, each mitochondrion was in close morphological relationship with a segment of the endoplasmic reticulum.

The mitochondria of developing mouse oocytes can be grouped into three types and are shown in Fig. 1. These mitochondria are: 1. those with no cristae (NC), 2. those containing typical cristae profiles (TC), and 3. mitochondria displaying the intraeristal vacuole (IV) described by YAMADA et al. (1957) and WISC~NITZ~R (1967). This micrograph is also exemplatory of the various relation- ships these organelles have to the cytoplasmic membrane system. All three types of mitochondria exhibit a close relationship to profiles of endoplasmic reticulum which can be either agranular or granular. Long strands of these membranes may be associated with more than one mitochondrion and, conversely, each mitochon- drion may have a close relationship to more than one segment of the reticulum (Fig. 1). The mitochondria may also be in apposition to relatively short profiles of the membrane system and with cytoplasmic vacuoles (Fig. 1).

The close relationship of these two organelles was a consistent observation in most oocytes studied during the time period covered by this investigation. This relationship has also been described in oocytes of hamster (WEAKLEY, 1967), guinea pig (ADAMS and HWRTIG, 1964), rabbit (ZAMBONI and MASTRGIAN~I, 1966), and human (HERTIG and ADAMS, 1967). A cursory examination of these mitochon- drial-membrane complexes is not particularly revealing. However, if one closely studies each of the mitochondria contained in every oocyte, it can often be seen tha t the cytoplasmic membrane system is continuous with the outer mitochondrial membrane. This continuity has been observed in various shapes and profiles. I t may appear as a long, tubular structure continuous with the outer membrane of the mitochondrion (Fig. 2) or, more typically, as a direct continuity with one of the long segments of endoplasmic reticulum (Fig. 3). Complexes similar to those described by ANDRE (1962) (i.e., fusion of the membrane system with the outer mitochondrial membrane) are often seen, but in some sections it is apparent tha t the cytoplasmic membrane is continuous with the outer mitoehondrial

Fig. 2. A tubular cytoplasmic membrane continuous with the outer mitochondrial membrane (arrows). • 148,000

Fig. 3. Profile of a cytoplasmic membrane continuous with the outer mitochondrial membrane (arrow). • 119,000

:Fig. 4. Cytoplasmic membrane continuous with the outer mitochondrial membrane (arrow). • 198,000

Fig. 5. Long profile of granular endoplasmic reticulum continuous with the outer mitochondrial membrane (arrow). • 126,000

3 z, Zellforsch., Bd. 97

F i g s . 2 - - 5

34 J . R . RUBY, R. F. DYEa, and R. G. SKALKO :

Figs. 6--8

Mitochondrial - - ER Continuities 35

Fig. 9. This micrograph demonstrates the connection of the rough endoplasmic reticulum with the outer mitochondrial membrane in an isolated mitochondrial preparation. • 121,000

membrane (Fig. 4). Long profiles of endoplasmic reticulum studded with ribo- somes are also directly continuous with the outer mitochondriai membrane (Fig. 5) and occasionally this type of continuity occurs at the dilated end of a cisternal space (Figs. 6, 7). A similar connection is shown in Fig. 8. In this instance, however, most of the reticular membrane is not visible but the conti- nuity of the triple-layered structure of the unit membrane between the mitochon- drion and endoplasmic reticulum is shown to advantage.

In the isolated preparations, contiguous and continuous relationships between granular reticulum and the outer mitochondrial membrane were routinely observed. An example of membrane continuities establishing an open channel of communication between the two organelles is depicted in Fig. 9.

Discussion This investigation demonstrates the continuity of endoplasmic reticulum

with outer mitochondrial membranes both in situ and in isolated preparations. This connection establishes a communication between the cisternal space of the

Fig. 6. An apparent connection between the dilated cisternal space of granular endoplasmic reticulum and a mitoehondrion (arrow). • 78,000

Fig. 7. Higher magnification of Fig. 6 demonstrating the continuity of the cytoplasmic membrane with the outer mitochondrial membrane (arrow). • 277,000

Fig. 8. Micrograph demonstrating continuity of the unit membrane of endoplasmic retieulum and outer mitochondrial membrane (arrows). x 110,000

3*

36 J .R. RuEY, R. F. DYER, and R. G. SKALKO :

cytoplasmic membrane system and the intracristal space of mitochondria. Previous observations of similar connections have been interpreted in two ways: 1. involvement in mitochondrial genesis (RoB]~RTSON, 1961; ANDRe, 1962) or 2. as channels through which materials may pass to and/or from mitochondria (BowMAN, 1967). On the basis of morphological evidence, it is difficult to ascertain which interpretation is correct, but the present investigation tends to support the latter viewpoint.

The continuities described by previous investigators (ROB]~RTSON, 1961; ANDRe, 1962; BOWMAN, 1967) are similar to the tubular membrane structure shown in Fig. 2 or resemble the configuration presented in Fig. 4. In the present study, these types of connections were the most obvious and were encountered frequently. Differences of opinion may arise regarding the functional significance of these connections when they are the only types observed. The possibility that some of these complexes are involved in mitochondrial genesis should not be overlooked. However, the observation tha t mitochondria are also continuous with ribosome-studded membranes would strongly suggest that these complexes are related to the transfer of proteins. This viewpoint is substantiated by several lines of evidence. First, the relationship of rough-surfaced endoplasmic reticulum to the synthesis and intracellular transport of proteins has been well established (PoRT~R, 1961 ; CARO and PALADE, 1964). Second, it has been reported tha t the only proteins synthesized by mitochondria are the insoluble structural proteins (TRUMAN, 1964; ROODYN, 1962). Mitochondria are unable to synthesize the soluble proteins (i.e., proteins of the major metabolic pathways) (KADENBACH, 1966; ROODYN etal., 1962; SIMPso~etal., 1961) and, in addition, it has been demonstrated that these soluble proteins are synthesized on the cytoplasmic membranes and subsequently transferred to the mitochondria (KAD~NBACH, 1966; HALDAR et al., 1967). The mechanism of this transfer has not been determined, but the current observation that an open channel exists between the two organelles suggests tha t this would be the preferred pathway for the transfer of proteins.

The fact that mitochondria from several tissues will accumulate ions against a gradient, both in vivo and in vitro [see LEHNINGER (1965) for review], suggests tha t Bowman's observation of connections between the tubular system and mitochondria in myocardium may also be related to protein transport rather than ion transfer as he suggested (BowMAN, 1967). Additionally, since mitochon- drial-endoplasmic reticulum complexes have been observed in several cell types (ROBERTSON, 1961; ANDRe, 1962; BOWMAN, 1967), it is plausible tha t these connections are universal among cells, but as yet have gone undetected.

Acknowledgements. This work was supported by grant number HD-03288 from the National Institutes of Child Health and Human Development, and Institutional Grants from the Louisiana State University Medical Center. We thank Doctors MELVIN HESS and ROBERT A. ME~ZIES for their advice during the preparation of this manuscript and Mrs. ELIZABETII ROUCHELL for her technical assistance.

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Doctor JOHN R. RUBY Department of Anatomy, Louisiana State University School of Medicine 1542 Tulane Avenue New Orleans, Louisiana 70112