Role of the Corpus Luteum and Progesterone in the Evolution of Vertebrate Viviparity

For the past 25 years we have used a comparative strategy designedto identify anddescribe the endocrine parameters of the oviparous-vivparoustransition and subsequent gradual reduction in hepatic yolkprotein precursor (vitellogenin) synthesis associated with placentalviviparity. Our approach has been to study vertebrate groupsin which both oviparous and viviparous modes are common (reptiles,elasmobranchs). We have provided evidence for the control offollicular (granulosa/theca) and luteal steroidogenesis, andthe cellular basis of gonadal steroid hormone action on thekey target tissues (oviduct, liver). Our results, some of whichare summarized below, have led us to suggest that ovarian progesterone(follicular or luteal in origin) has a dual role in the evolutionof viviparity: 1. To inhibit myometrial contractions, thus providinga primary condition for egg retention and viviparity. 2. Toinhibit estrogen-induced hepatic vitellogenin synthesis as partof both normal oviparous cycles and as a concomitant of placentalevolution.     

The role of steroids in reproduction in female elasmobranchs and reptiles

Adequate evidence exists to suggest the importance of temporal changes in steroid hormone ratios in the normal reproductive/vitellogenin cycle in oviparous and viviparous elasmobranchs and reptiles. In oviparous species, where the cycle is relatively short, secretion of gonadal hormones is synchronous; thus inhibitory actions of progesterone (P) on hepatic or reproductive tract functions would be offset by stimulatory actions of estradiol (E), resulting in appropriate vitellogenin secretion and reproductive tract development. In viviparous species, temporal asynchrony of E and P secretion occurs, and the actions of the individual hormones can be more easily dissected out. Thus, during gestation, where P is the dominant hormone, antagonistic or stimulatory actions of E may be prevented, and the inhibitory action of P on vitellogensis dominant. Hence vitellogenesis is limited to the follicular phase and eggs are retained.

Although the elasmobranch and reptilian species discussed here do not form a continuum through phylogenesis, but rather are extant forms of a particular line of evolution, it is possible to extrapolate from these observations to the probable endocrine interactions in a species as viviparity evolves from oviparity. The theoretical intermediate stage would involve; (a) egg retention, (b) extension of the luteal phase and increased P secretion and (c) resulting in E/P asynchrony and potential expression of “independent” P action, egg retention and yolk suppression.     

Ovarian steroid synthesis and the hormonal control of the elasmobranch reproductive tract

Synopsis The reproductive biology of the chondricthyan fishes is remarkably sophisticated. Using both oviparous and viviparous reproductive modes, the group has generally adapted the style of bringing forth relatively few young at one time, each representing the investment of a great deal of maternal energy. The oviparous species foreshadow the situation common in oviparous reptiles and universal in birds. On the other hand, viviparous species range from simple internal incubators, in which large yolked eggs are retained, to other species in which the complexity of placentation and yolk reduction approach the eutherian condition. Further, in certain viviparous elasmobranchs the phenomenon of histotrophic nutrition attains an importance and complexity not seen in any other vertebrate group including mammals. Internal fertilization and amniote patterns of reproductive tract development also operate in virtually all elasmobranchs. The summary of work presented here suggests that these female reproductive styles are associated with a reproductive endocrinology which is the archetype for amniote vertebrates.     

Behavioral indices of estrus in a group of captive African elephants (Loxodonta africana)

This study investigated behavioral signals of estrus by systematically monitoring the interactions of one male with four female African elephants housed in a naturalistic outdoor enclosure at Disney's Animal Kingdom over a period of 11 months. We measured changes in five spatial behaviors and 22 tactile‐contact behaviors, as well as changes in serum progestagen and LH concentrations, across three ovarian cycles for each female. Two females did not cycle during the study. Three different phases of the ovarian cycle were identified: mid luteal, anovulatory follicular, ovulatory follicular. The male followed more and carried out more genital inspections, flehmen, and trunk‐to‐mouth behaviors toward cycling females during their ovulatory phase. Genital inspections by the male peaked above baseline levels on the day of an LH surge, and up to 9 days before, in both cycling females and, thus, might be a useful behavioral index of estrus. The male also carried out more genital inspections, flehmen, and trunk touches to the back leg toward ovulatory cycling than noncycling females. Overall, our results indicated that: 1) a single subadult African elephant male could discriminate two females in the ovulatory phase of their cycle (i.e., during the 3 weeks preceding ovulation) from the mid luteal phase; 2) the male also discriminated two cycling females in the ovulatory and anovulatory follicular phases from two noncycling females; 3) two females in the ovulatory phase of the cycle displayed a greater variety of tactile‐contact behavior toward the male compared to the other cycle phases. Zoo Biol 0:1–19, 2005. © 2005 Wiley‐Liss, Inc.     

From oviparity to viviparity: a preliminary note on the morphometric differentiation between oviparous and viviparous species assigned to the genus Liolaemus (Reptilia, Squamata, Liolaemidae)

Embryonic growth requires a considerable internal space in viviparous female lizards and this need for space should be reflected in their external morphometry. External morphological differences associated with the reproductive mode in 12 viviparous and 18 oviparous species of Liolaemus lizards were identified. Size differences between viviparous and oviparous species were elucidated by axilla-groin/snout-vent relationship. Axilla-groin distance, considered a size estimator of visceral cavity, surpassed 50% of snout-vent length in viviparous females, while it is always less than 50% in oviparous females. This difference between the two reproductive modes is statistically significant.     

Investigation of individual and group variability in estrous cycle characteristics in female Asian elephants (Elephas maximus) at the Oregon Zoo

Evaluating ovarian cycle activity through longitudinal progestagen monitoring is important for optimizing breeding management of captive elephants and understanding impact of life events (births, deaths, and transfers) on reproductive function. This study summarized serum progestagen profiles for eight Asian mainland elephants (Elephas maximus indicus) and one Bornean elephant (E. maximus borneensis) at the Oregon Zoo over a 20-yr interval, and represents the longest longitudinal dataset evaluated to date. Estrous cycle characteristics were more varied than previously reported for this species, with an overall duration of 12 to 19 wk, luteal phase duration of 4 to 15 wk, and follicular phase duration of 2 to 12 wk. In general, there was more cycle variability across than within individual elephants. Compared with other elephants in the group, the Borneo female exhibited consistently longer cycle lengths, higher progestagen concentrations, and greater cycle variability; however, it is not known if this represents a subspecies or an individual difference. Cycle durations did not appear to change over time or with age, and the first pubertal cycle was similar to subsequent cycles. Variability in duration of the follicular phase was greater than that of the luteal phase. In addition, there was a significant negative relationship between luteal and follicular phase durations, suggesting a possible regulatory role of the follicular phase in maintaining a relatively consistent cycle duration within individuals. Overall, we found these elephants to be highly resilient in that major life events (births, deaths, and changes in herd structure) had minimal effect on cycle dynamics over time. In conclusion, the higher range in cycle phase characteristics is likely because of the larger number of elephants studied and longer duration of longitudinal monitoring, and may be more representative of the captive population as a whole. Furthermore, identification of significant interanimal variability suggests that understanding the complexities of herd reproductive characteristics could facilitate development of more effective institution-specific breeding management strategies.     

Reproductive cycles in sheep

During the last three decades, there has been remarkable progress in many aspects of ovarian biology due to advances in real-time ultrasonography, which permits non-invasive, repeated monitoring of ovarian structures in conscious and non-anaesthetised animals. This review is primarily concerned with ovarian activity, as determined by transrectal ultrasonography, and measurements of circulating concentrations of gonadotrophins and ovarian steroids during reproductive cycles in sheep. The growth of antral follicles reaching ostensibly ovulatory sizes occurs in a wave-like pattern throughout the breeding season in both prolific and non-prolific breeds of sheep. There are typically 3 or 4 waves of follicle development during the interovulatory interval. Follicular wave emergence is primarily controlled by changes in circulating concentrations of follicle-stimulating hormone (FSH) but diminished ovarian responsiveness to gonadotrophic signals may result in reduced numbers of follicular waves. In cyclic ewes, the largest ovarian follicles acquire the ability to secrete oestradiol from the day of emergence with peak oestradiol secretion occurring about the time they reach maximum diameter. The high ovulation rate in some prolific breeds may be achieved by the ovulation of follicles from the last two waves of the interovulatory interval. Prolific ewes tend to produce more but smaller corpora lutea (CL) and have lower serum concentrations of progesterone during the luteal phase of the oestrous cycle as compared to less prolific genotypes. Lastly, recent studies of the endocrine influences on ovarian function have brought into question the existence of strong follicular dominance, as seen in cattle, and provided new insights into the effects of luteal progesterone on antral follicular development in ewes.     

Mononuclear cell-modulation of granulosa cell steroidogenesis in rat: influence of ovarian cycle.

The mononuclear cells exert a paracrine influence on the ovarian function, including steroidogenesis. This study examines the ability of the conditioned medium from the cultures of splenic mononuclear cells, obtained during various phases of the ovarian cycle, on progesterone accumulation by the granulosa cells in the culture medium. Female Wistar rats, aged twenty-five days, were made pseudopregnant by an injection of pregnant mare's serum gonadotropin. The splenic mononuclear cells were isolated at follicular phase, early luteal phase, mid luteal phase and late luteal phase and cultured for 48 h. The ammonium sulphate precipitated fraction of the conditioned medium was added to the granulosa cells obtained from immature rats treated with diethylstillboestrol. The granulosa cells were cultured for 48 h, and the progesterone accumulated in the medium was assayed. The conditioned medium from the cultures of the mononuclear cells obtained during follicular phase and late luteal phase inhibited FSH-induced progesterone secretion, whereas conditioned medium obtained from mid luteal phase mononuclear cells enhanced the effect of FSH. The stimulatory effect of db-cAMP on progesterone accumulation in the culture medium is inhibited by conditioned medium obtained from all the phases of the ovarian cycle. This study demonstrates a cyclicity in the behaviour of the splenic mononuclear cells on ovarian steroidogenesis, suggesting a bi-directional paracrine and/or endocrine relationship between ovary and the mononuclear cells.     

Effects of endocrine disruptors on reproduction in viviparous teleosts with intraluminal gestation

Many water systems worldwide are affected by pollutants, including potential endocrine disruptors (EDs). Most studies on the effects of EDs on fish reproduction have focused on oviparous species. However, some important groups of fishes are not oviparous and there is scarce information about how EDs affect species with alternative reproductive modes. Goodeinae is a viviparous matrotrophic subfamily with intraluminal gestation (IG), where transfer of nutrients occurs and embryos develop inside the ovarian cavity. Goodeinae is endemic to the Mexican Central Plateau, an area affected by potential EDs, including 2,4-dichlorophenoxyacetic acid (2,4-D). This review synthesizes the available information about EDs in viviparous teleosts with IG and performs a case study on the effects of 2,4-D on gonadal structure of two Goodeinae species. We hypothesized that individuals exposed to 2,4-D might show altered gonadal structure. The available information included effects on gene expression, sexual differentiation, gametogenesis, secondary sexual characteristics, and sexual behavior. Knowledge gaps persisted on the effects of EDs on viviparous teleosts with IG. Holistic approaches are needed to understand the mechanisms underlying endocrine disruption effects. Field studies are needed to evaluate the consequences of EDs on wild populations. The case study revealed histological alterations in oocytes, spermatogonia, and sperm cysts in fishes exposed to 2,4-D. Ultrastructurally, gonads exhibited alterations in oocyte mitochondrial and nuclear membranes, and in spermatid mitochondria. The observed changes could be related to 2,4-D exposure, which may affect species reproduction in their natural environment. Matrotrophic viviparous teleosts with IG may serve as models to explore endocrine disruption.     

Female body odour is a potential cue to ovulation

Human body odours have been reported to influence female mate choice. Women prefer the odours of immunocompatible men and, during their fertile period, judge the body odours of men with symmetrical bodies--which is indicative of genetic quality--as sexy and pleasant. The reproductive success of men largely depends on mating with fertile women, but it is not known whether men can detect a woman's fertile period. We asked women who had regular menstrual cycles and who were not using hormonal contraceptives to wear a T-shirt for three consecutive nights during their late follicular (ovulatory) phase and another T-shirt during the luteal (non-ovulatory) phase of their menstrual cycle. Male raters judged the odours of T-shirts worn during the follicular phase as more pleasant and sexy than odours from T-shirts worn during the luteal phase. The odour differences between the follicular and luteal phases did not dissipate quickly over time as male raters were able to detect and judge follicular phase body odours as more pleasant and sexy than the odours from the luteal phase even after the T-shirts were kept at room temperature for one week. These findings suggest that ovulation may not be concealed and that men could use ovulation-linked odours in their mate selection.     

Experimental evidence of early costs of reproduction in conspecific viviparous and oviparous lizards

Reproduction entails costs, and disentangling the relative importance of each stage of the reproductive cycle may be important to assess the costs and benefits of different reproductive strategies. We studied the early costs of reproduction in oviparous and viviparous lizard females of the bimodal reproductive species Zootoca vivipara. Egg retention time in oviparous females is approximately one-third of the time in viviparous females. We compared the vitellogenesis and egg retention stages that are common to both reproductive modes. Precisely, we monitored the thermoregulatory behaviour, the weight gain and the immunocompetence of the females. Moreover, we injected an antigen in half of the females (immune challenge) to study the trade-offs between reproductive mode and immune performance and between different components of the immune system. Finally, we experimentally induced parturition in viviparous females at the time of egg laying in oviparous females. Oviparous and viviparous females did not show strong differences in response to the immune challenge. However, viviparous females spent more time thermoregulating while partially hidden and gained more weight than oviparous females. The greater weight gain indicates that the initial period of egg retention is less costly for viviparous than for oviparous females or that viviparous females are able to save and accumulate energy at this period. This energy may be used by viviparous females to cope with the subsequent costs of the last two-third of the gestation. Such an ability to compensate the higher costs of a longer egg retention period may account for the frequent evolution of viviparity in squamate reptiles.     

Seasonal effects on the endocrine pattern of semi-captive female Asian elephants (Elephas maximus): timing of the anovulatory luteinizing hormone surge determines the length of the estrous cycle

Better breeding strategies for captive Asian elephants in range countries are needed to increase populations; this requires a thorough understanding of their reproductive physiology and factors affecting ovarian activity. Weekly blood samples were collected for 3.9 years from 22 semi-captive female Asian elephants in Thai elephant camps to characterize LH and progestin patterns throughout the estrous cycle. The duration of the estrous cycle was 14.6+/-0.2 weeks (mean+/-S.E.M.; n=71), with follicular and luteal phases of 6.1+/-0.2 and 8.5+/-0.2 weeks, respectively. Season had no significant effect on the overall length of the estrous cycle. However, follicular and luteal phase lengths varied among seasons and were negatively correlated (r=-0.658; P<0.01). During the follicular phase, the interval between the decrease in progestin concentrations to baseline and the anovulatory LH (anLH) surge varied in duration (average 25.9+/-2.0 days, range 7-41, n=23), and was longer in the rainy season (33.4+/-1.8 days, n=10) than in both the winter (22.2+/-4.5 days, n=5; P<0.05) and summer (18.9+/-2.6 days, n=8; P<0.05). By contrast, the interval between the anLH and ovulatory LH (ovLH) surge was more consistent (19.0+/-0.1 days, range 18-20, n=14). Thus, seasonal variation in estrous cycle characteristics were mediated by endocrine events during the early follicular phase, specifically related to timing of the anLH surge. Overall reproductive hormone patterns in Thai camp elephants were not markedly different from those in western zoos. However, this study was the first to more closely examine how timing of the LH surges impacted estrous cycle length in Asian elephants. These findings, and the ability to monitor reproductive hormones in range countries (and potentially in the field), should improve breeding management of captive and semi-wild elephants.     

Faecal steroid metabolites for non-invasive assessment of reproduction in common warthogs (Phacochoerus africanus), red river hogs (Potamochoerus porcus) and babirusa (Babyrousa babyrussa)

The objectives of this study were to analyse faecal steroid metabolites in African and South East Asian pig species kept in European zoos. Species studied were the warthog (Phacochoerus africanus), the red river hog (Potamochoerus porcus) and the babirusa (Babyrousa babyrussa). Faecal samples were collected 1-3 times per week from non-pregnant and pregnant captive female warthogs (n = 9), red river hogs (n = 7) and babirusas (n = 5). Enzyme-immunoassays for faecal progesterone, androgen, and oestrogen metabolites, were tested for their ability to determine follicular and luteal phases. In all three species, oestrous cycles could be monitored with 20alpha-OH- and 20-oxo-pregnane assays. In contrast, oestrogens and androgens were not useful in characterising follicular activity during the oestrous cycle in any species. Faecal 20alpha-OH- and 20-oxo-pregnane values were significantly correlated. Faecal pregnane concentrations revealed species-specific differences. Luteal phase values of 20alpha-OH-pregnanes were considerably higher than 20-oxo-pregnanes; 20alpha-OH-pregnanes were in the range of 3-10 microg/g in warthogs and red river hogs, whereas concentrations were 30-200 microg/g faeces in the babirusa. Regular oestrus cycles had a length of about 35 days in all three species studied. Results indicated a seasonal influence on the occurrence of reproductive cycles in the warthog with anoestrous periods in the European summer. The red river hog was found to be a seasonal and poly oestrous breeder; oestrus cycles started by January and continued until summer. In contrast, the babirusa showed non-seasonal ovarian cyclicity. In pregnant red river hogs, progesterone metabolites were comparable to luteal phase values of the oestrous cycle during the first 3 months of gestation, but did further increase during the last month of pregnancy. Oestrogens and 17-oxo-androstanes were significantly elevated during the second half of gestation. In summary, the reproductive biology of three exotic pig species was studied using non-invasive faecal steroid analysis and these methods were used for comparative investigations of oestrous cycles, pregnancy and seasonality.     

A phylogenetic analysis of variation in reproductive mode within an Australian lizard (Saiphos equalis, Scincidae)

Saiphos equalis , a semi-fossorial scincid lizard from south-eastern Australia, is one of only three reptile species world-wide that are known to display geographic variation in reproductive mode. Uniquely, Saiphos equalis includes populations with three reproductive modes: oviparous with long (15-day) incubation periods; oviparous with short (5-day) incubation periods; and viviparous (0-day incubation periods). No Saiphos populations show 'normal' scincid oviparity (> 30-day incubation period). We used mitochondrial nucleotide sequences ( ND2 and cytochrome b ) to reconstruct relationships among populations from throughout the species' distribution in New South Wales, Australia. Under the phylogenetic species concept, phylogenetic analyses are consistent with the oviparous and viviparous populations of S. equalis being conspecific. Phylogenetic analyses suggest that the long incubation period oviparous lineage is the sister group to all other populations; and that the viviparous populations belong to a cluster of weakly supported clades basal to the short-incubation-period oviparous clade. These clades correspond to variation in reproductive mode and geographic location.     

Relationship between ovarian cycle phase and sexual behavior in female Japanese macaques (Macaca fuscata)

We conducted behavioral observations simultaneously with fecal sample collection on eight nonlactating females 2-3 times per week, October 1997-March 1998, to examine the relationship between ovarian hormones and the sexual behavior of female Japanese macaques (Macaca fuscata) during the mating season. We analyzed samples by enzyme immunoassay for fecal hormone levels. Hormone profiles of estrone-glucuronide (E1) and pregnanediol-glucuronide (PdG) were used to separate ovarian cycles into three phases (follicular, periovulatory, and luteal). Hormonal profiles indicate average cycle lengths of 27.6 +/- 4.2 days (+/- SD; n = 26). Average lengths of the luteal and follicular phases were 12.3 +/- 3.8 days (+/- SD) and 8.3 +/- 3.4 days (+/- SD), respectively. We observed female Japanese macaques engaging in sexual activity throughout the ovarian cycle, with the highest rates occurring during the follicular and periovulatory phases as compared to the luteal phase. The attractivity of female Japanese macaques increased significantly during the follicular and periovulatory phases of the ovarian cycle, when E1 levels are peaking and PdG levels drop to baseline. In addition, females displayed a significant increase in proceptive behavior during the follicular and periovulatory phases. Grooming bouts, as well as proximity between female and male macaques, also increased significantly during the follicular and periovulatory phases. We conclude that fluctuating levels of ovarian hormones in different phases of the cycle are significantly associated with variable rates of copulatory and pericopulatory behaviors in these Japanese macaque females.     

Follicular waves during the oestrous cycle in Nili-Ravi buffaloes undergoing spontaneous and PGF2alpha-induced luteolysis

The objective of this study was to characterize the follicular waves and associated ovarian events during spontaneous and PGF(2alpha)-induced oestrous cycles in Nili-Ravi buffaloes. In Exp. 1, (n=13 oestrous cycles) follicular and luteal development was monitored by ultrasonography and jugular blood samples were collected simultaneously on alternate days. Of 12 oestrous cycles, 9 (75%) had two waves of follicular activity and only 3 (25%) had three waves. The mean (+/-S.E.M.) length of the oestrous cycle was shorter (P<0.05) in buffaloes with two waves than in those with three waves (21.2+/-0.1 days versus 22.8+/-0.1 days). In Exp. 2, follicular dynamics were compared in buffaloes undergoing spontaneous (n=12 oestrous cycles) and PGF(2alpha)-induced (n=9) regression of the corpus luteum (CL). The dynamics of ovulatory follicular growth during the 3 days before oestrus were similar (P>0.05) in buffaloes undergoing spontaneous and PGF(2alpha)-induced luteolysis. These results show that (1) the majority of buffaloes had a two wave pattern of follicular growth and emergence of a third wave was associated with a longer luteal phase, and (2) follicular dynamics during the 3 days before oestrus were similar in buffaloes undergoing spontaneous and PGF(2alpha)-induced luteolysis.     

Lipid class composition of the viviparous yellowtail rockfish over a reproductive cycle

Triacylglycerols (TAG) were the dominant lipids in liver tissue of both sexes of the viviparous yellowtail rockfish Sebastes flavidus during the spring and summer when greatest feeding occurred. Significant declines in liver TAG during the winter corresponded to increased concentrations of polar lipids (PL), the main component of cell membranes. Elevated PL in female livers relative to those of males preceded the period of ovarian enlargement and may be attributed to the production of vitellogenin. During late vitellogenesis and gestation, ovaries contained significantly elevated concentrations of PL, TAG, and cholesterol. Since yellowtail rockfish are highly fecund and viviparous, large quantities of ovarian PL are required for proliferation of the cell membranes in developing larval tissues. TAG accumulated in ovaries in lower concentrations than PL and were likely metabolized as the main source of energy during gestation. This pattern of ovarian PL concentrations exceeding those of TAG and the presence of oil globules, may be unique to highly fecund, viviparous teleosts and signify an alternative profile to those previously documented for oviparous species. Testes at maximum I _G were only 7% of maximum ovarian size, composed of mainly PL, and lacked a defined lipid dynamic pattern across the reproductive cycle.     

Proliferation of Oogonia and folliculogenesis in the viviparous teleost Ilyodon whitei (Goodeidae)

Oogonial proliferation in fishes is an essential reproductive strategy to generate new ovarian follicles and is the basis for unlimited oogenesis. The reproductive cycle in viviparous teleosts, besides oogenesis, involves development of embryos inside the ovary, that is, intraovarian gestation. Oogonia are located in the germinal epithelium of the ovary. The germinal epithelium is the surface of ovarian lamellae and, therefore, borders the ovarian lumen. However, activity and seasonality of the germinal epithelium have not been described in any viviparous teleost species regarding oogonial proliferation and folliculogenesis. The goal of this study is to identify the histological features of oogonial proliferation and folliculogenesis during the reproductive cycle of the viviparous goodeid Ilyodon whitei. Ovaries during nongestation and early and late gestation were analyzed. Oogonial proliferation and folliculogenesis in I. whitei, where intraovarian gestation follows the maturation and fertilization of oocytes, do not correspond to the late oogenesis, as was observed in oviparous species, but correspond to late gestation. This observation offers an example of ovarian physiology correlated with viviparous reproduction and provides elements for understanding the regulation of the initiation of processes that ultimately result in the origin of the next generation. These processes include oogonia proliferation and development of the next batch of germ cells into the complex process of intraovarian gestation. J. Morphol. 275:1004–1015, 2014. © 2014 Wiley Periodicals, Inc.     

Development of the uterine shell glands during the preovulatory and early gestation periods in oviparous and viviparous Lacerta vivipara

The evolutionary process leading to the emergence of viviparity in Squamata consists of lengthening the period of egg retention in utero coupled with marked reduction in the thickness of the eggshell. We used light microscopy and scanning electron microscopy to study uterine structure during the reproductive cycle of oviparous and viviparous females of the reproductively bimodal Lacerta vivipara. We compared the structure of the uterine shell glands, which secrete components of the eggshell, during preovulatory and early gestation phases of the reproductive cycle and also compared histochemistry of the eggshells. The uterine glands of both reproductive forms undergo considerable growth within a period of a few weeks during folliculogenesis and vitellogenesis preceding ovulation. The majority of the proteinaceous fibers of the shell membrane are secreted early in embryonic development and the uterine glands regress shortly thereafter. This supports previous observations indicating that, in Squamata, secretion of the shell membrane occurs very rapidly after ovulation. The most striking differences between reproductive modes were larger uterine glands at late vitellogenesis in oviparous females, 101 microm compared to 60 microm in viviparous females, and greater thickness of the shell membrane during early gestation in oviparous females (52-73 microm) compared to viviparous females (4-8 microm). Our intraspecific comparison supports the conclusions of previous studies that, prior to ovulation, the uterine glandular layer is less developed in viviparous than in oviparous species, and that this is the main factor accounting for differences in the thickness of the shell membrane of the two reproductive forms of squamates.