Neonatal Ewe Research Articles

Progesterone Inhibits Uterine Gland Development in the Neonatal Mouse Uterus1

Uterine glands and their secretions are required for conceptus (embryo/fetus and associated placenta) survival and development. In most mammals, uterine gland morphogenesis or adenogenesis is a uniquely postnatal event; however, little is known about the mechanisms governing the developmental event. In sheep, progestin treatment of neonatal ewes permanently ablated differentiation of the endometrial glands. Similarly, progesterone (P4) inhibits adenogenesis in neonatal mouse uterus. Thus, P4 can be used as a tool to discover mechanisms regulating endometrial adenogenesis. Female pups were treated with sesame vehicle alone as a control or P4 from Postnatal Day 2 (PD 2) to PD 10, and reproductive tracts were examined on PD 5, 10, or 20. Endometrial glands were fully developed in control mice by PD 20 but not in P4-treated mice. All other uterine cell types appeared normal. Treatment with P4 stimulated proliferation of the stroma but suppressed proliferation of the luminal epithelium. Microarray analysis revealed that expression of genes were reduced (Car2, Fgf7, Fgfr2, Foxa2, Fzd10, Met, Mmp7, Msx1, Msx2, Wnt4, Wnt7a, Wnt16) and increased (Hgf, Ihh, Wnt11) by P4 in the neonatal uterus. These results support the idea that P4 inhibits endometrial adenogenesis in the developing neonatal uterus by altering expression of morphoregulatory genes and consequently disrupting normal patterns of cell proliferation and development.

Postnatal uterine development in Inverdale ewe lambs

Postnatal development of the uterus involves, particularly, development of uterine glands. Studies with ovariectomized ewe lambs demonstrated a role for ovaries in uterine growth and endometrial gland development between postnatal days (PNDs) 14 and 56. The uterotrophic ovarian factor(s) is presumably derived from the large numbers of growing follicles in the neonatal ovary present after PND 14. The Inverdale gene mutation (FecXI) results in an increased ovulation rate in heterozygous ewes; however, homozygous ewes (II) are infertile and have 'streak' ovaries that lack normal developing of preantral and antral follicles. Uteri were obtained on PND 56 to determine whether postnatal uterine development differs between wild-type (++) and II Inverdale ewes. When compared with wild-type ewes, uterine weight of II ewes was 52% lower, and uterine horn length tended to be shorter, resulting in a 68% reduction in uterine weight:length ratio in II ewes. Histomorphometrical analyses determined that endometria and myometria of II ewes were thinner and intercaruncular endometrium contained 38% fewer endometrial glands. Concentrations of estradiol in the neonatal ewes were low and not different between ++ and II ewes, but II ewes had lower concentrations of testosterone and inhibin-alpha between PNDs 14 and 56. Receptors for androgen and activin were detected in the neonatal uteri of both ++ and II ewes. These results support the concept that developing preantral and/or antral follicles of the ovary secrete uterotrophic factors, perhaps testosterone or inhibin-alpha, that acts in an endocrine manner to stimulate uterine growth and endometrial gland development in the neonatal ewes.

POSTNATAL UTERINE DEVELOPMENT IN INVERDALE EWE LAMBS

Postnatal development of the uterus involves, particularly, development of uterine glands. Studies with ovariectomized ewe lambs demonstrated a role for ovaries in uterine growth and endometrial gland development between postnatal days (PNDs) 14 and 56. The uterotrophic ovarian factor(s) is presumably derived from the large numbers of growing follicles in the neonatal ovary present after PND 14. The Inverdale gene mutation (FecXI) results in an increased ovulation rate in heterozygous ewes; however, homozygous ewes (II) are infertile and have 'streak' ovaries that lack normal developing of preantral and antral follicles. Uteri were obtained on PND 56 to determine whether postnatal uterine development differs between wild-type (++) and II Inverdale ewes. When compared with wild-type ewes, uterine weight of II ewes was 52% lower, and uterine horn length tended to be shorter, resulting in a 68% reduction in uterine weight:length ratio in II ewes. Histomorphometrical analyses determined that endometria and myometria of II ewes were thinner and intercaruncular endometrium contained 38% fewer endometrial glands. Concentrations of estradiol in the neonatal ewes were low and not different between ++ and II ewes, but II ewes had lower concentrations of testosterone and inhibin-alpha between PNDs 14 and 56. Receptors for androgen and activin were detected in the neonatal uteri of both ++ and II ewes. These results support the concept that developing preantral and/or antral follicles of the ovary secrete uterotrophic factors, perhaps testosterone or inhibin-alpha, that acts in an endocrine manner to stimulate uterine growth and endometrial gland development in the neonatal ewes.

WNT Pathways in the Neonatal Ovine Uterus: Potential Specification of Endometrial Gland Morphogenesis by SFRP21

Endometrial glands are critical for uterine function and develop between birth (Postnatal Day [P] 0) and P56 in the neonatal ewe. Endometrial gland morphogenesis or adenogenesis involves the site-specific budding differentiation of the glandular epithelium from the luminal epithelium followed by their coiling/branching development within the stroma of the intercaruncular areas of the endometrium. To determine whether WNT signaling regulates endometrial adenogenesis, the WNT signaling system was studied in the neonatal ovine uterus. WNT5A, WNT7A, and WNT11 were expressed in the uterine epithelia, whereas WNT2B was in the stroma. The WNT receptors FZD2 and FZD6 and coreceptor LRP6 were detected in all uterine cells, and FZD6 was particularly abundant in the endometrial epithelia. Secreted FZD-related protein-2 (SFRP2), a WNT antagonist, was not detected in the P0 uterus, but was abundant in the aglandular caruncular areas of the endometrium between P7 and P56. Exposure of ewes to estrogens during critical developmental periods inhibits or retards endometrial adenogenesis. Estrogen-induced disruption of endometrial adenogenesis was associated with reduction or ablation of WNT2B, WNT7A, and WNT11, and with an increase in WNT2 and SFRP2 mRNA, depending on exposure period. Collectively, results implicate the canonical and noncanonical WNT pathways in regulation of postnatal ovine uterine development and endometrial adenogenesis. Expression of SFRP2 in aglandular caruncular areas may inhibit the WNT signaling pathway, thereby concentrating WNT signaling and restricting endometrial adenogenesis in the intercaruncular areas of the uterus. Further, estrogen-induced inhibition of adenogenesis may be mediated by a reduction in WNT signaling caused by aberrant induction of SFRP2 and loss of several critical WNTs.

The IGF system in the neonatal ovine uterus

Postnatal development of the ovine uterus primarily involves uterine gland morphogenesis or adenogenesis. Adenogenesis involves the budding differentiation of the glandular epithelium (GE) from the luminal epithelium (LE) and then GE proliferation and coiling/branching morphogenetic development within the stroma between birth (postnatal day or PND 0) and PND 56. Insulin-like growth factor (IGF)-I and IGF-II mRNAs were previously found to be expressed only in the endometrial stroma, whereas the IGF receptor (IGF-1R) mRNA was most abundant in epithelia and in stroma, suggesting that an intrinsic IGF system regulates postnatal development of the uterus. Given that the biological activities of IGFs are modulated by a family of six IGF binding proteins (IGFBPs) and specific proteases, the objective was to determine the effects of age and estrogen disruption on expression of IGFs, IGFBPs and pregnancy-associated plasma protein A (PAPP-A or IGFBP-4 protease) in the ovine uterus. In Study One, circulating levels of IGF-I and IGF-II in the serum of neonatal ewes did not change between PND 0 and PND 56. Levels of immunoreactive IGF-I, IGF-II and IGF-1R protein were most abundant on the apical surface of the endometrial LE and GE. RT-PCR analyses detected expression of IGFBPs (3, 4, 5 and 6) as well as PAPP-A mRNAs in the uterus, but not IGFBP-1 and IGFBP-2 mRNAs. IGFBP-3 and IGFBP-4 mRNAs were expressed specifically in the endometrial stroma and myometrium and increased after birth. PAPP-A mRNA was expressed specifically in the endometrial stroma and increased after birth. In Study Two, ewes were treated from birth with estradiol-17beta valerate (EV), which reduces uterine growth and inhibits endometrial adenogenesis. On PNDs 14 and 56, IGFBP-3 mRNA was decreased in the uterus of EV-treated ewes, but IGF-1R and IGFBP-4 mRNAs were not affected. PAPP-A mRNA was increased by EV treatment on PND 14, but decreased on PND 56. These results support the hypothesis that an intrinsic IGF system in the uterus regulates epithelial-stromal interactions important for postnatal uterine growth and endometrial gland morphogenesis in the sheep.

Neonatal estrogen exposure disrupts uterine development in the postnatal sheep.

Postnatal development of the ovine uterus between birth and postnatal day (PND) 56 involves budding differentiation of the endometrial glandular epithelium from the luminal epithelium (LE) followed by extensive coiling and branching morphogenesis of the tubular glands. To determine the short- and long-term effects of estrogen on neonatal ovine uterine development after PND 14, neonatal sheep were randomly assigned at birth (PND 0) to be treated daily with estradiol-17beta benzoate (EB; 0, 0.01, 0.1, 1, or 10 microg/kg body weight.d) during one of two developmental periods (PND 14-27 or 42-55). All ewes were hemiovariohysterectomized at the end of EB treatment on either PND 28 or 56, and the remaining uterine horn and ovary removed on PND 112. Immediate responses to EB treatment included dose- and age-dependent increases in uterine wet weight, thickness of the endometrium, myometrium, and LE, but decreases in endometrial glands on PND 28 and 56. Transient exposure to EB decreased gland number and thickness of the endometrium and LE on PND 112 but did not affect extrauterine reproductive tract structures. The mechanism of estrogen inhibition of uterine development did not involve effects on cell proliferation. Real-time PCR analyses found that EB exposure disrupted normal patterns of growth factor (IGF-I, IGF-II, fibroblast growth factor-7, fibroblast growth factor-10, and hepatocyte growth factor) and receptor mRNA expression in the uterus. Transient exposure of the neonatal ewe to estrogens during critical periods specifically alters growth factor networks that perturb normal development of the uterus, leading to permanent alterations in uterine structure and function.

003.Uterine and placental factors regulating conceptus growth: insights from the ewe

Uterine adenogenesis is the process whereby endometrial glands differentiate and develop and is primarily a postnatal event in all mammals. In domestic animals and humans, adenogenesis involves initial differentiation and budding of glandular epithelium followed by invagination and extensive tubular coiling and branching morphogenesis through the endometrial stroma to the myometrium. Transient exposure of the neonatal ewe to a progestin from birth to postnatal Day 56 resulted in a uterine gland knock out (UGKO) phenotype in the adult. UGKO ewes exhibit a peri-implantation defect in conceptus (embryo/fetus and associated extraembryonic membranes) survival, indicating the functional importance of uterine glands and their secretions. Genomic and proteomic analysis of uterine endometrium from UGKO ewes has identified many candidate genes that regulate conceptus development and implantation, including endogenous Jaagsiekte sheep retroviruses (enJSRVs), glycosylated cell adhesion molecule one (GlyCAM-1), osteopontin and galectin-15. Galectin-15, also known as OVGAL11, and a previously uncharacterised member of the galectin family of secreted β-galactoside lectins, was discovered in the endometrium of sheep. In endometria of cyclic and pregnant sheep, galectin-15 mRNA was expressed specifically in the endometrial luminal epithelium but not in the conceptus. In pregnant sheep, galectin-15 mRNA expression appeared in the epithelia between Days 10 and 12 and increased between Days 12 and 16. Progesterone induced and interferon tau stimulated galectin-15 mRNA in the endometrial epithelium. Galectin-15 protein was concentrated near and on the apical surface of the endometrial luminal epithelia and localised within discrete cytoplasmic crystalline structures of conceptus trophectoderm. Galectin-15 is hypothesised to function extracellularly to regulate trophectoderm migration and adhesion to the endometrial epithelium and intracellularly to regulate cell survival, growth and differentiation. In sheep, the sequential actions of ovarian steroid hormones (oestrogen and progesterone), interferon tau, placental lactogen and placental growth hormone constitute a servomechanism that directly regulates endometrial gland morphogenesis and terminal differentiated function to provide increasing histotrophic nutrition for conceptus growth and development. Knowledge gained from this research will be used to prevent or treat infertility, fetal growth retardation and disease in domestic animals and humans.

Estrogen and antiestrogen effects on neonatal ovine uterine development.

Postnatal development of the ovine uterus between birth and Postnatal Day (PND) 56 involves differentiation of the endometrial glandular epithelium from the luminal epithelium followed by tubulogenesis and branching morphogenesis. These critical events coincide with expression of estrogen receptor alpha (ERalpha) by nascent endometrial glands and stroma. To test the working hypothesis that estrogen and uterine ERalpha regulate uterine growth and endometrial gland morphogenesis in the neonatal ewe, ewes were treated daily from birth (PND 0) to PND 55 with 1) saline and corn oil as a vehicle control (CX), 2) estradiol-17 beta (E2) valerate (EV), an ERalpha agonist, 3) EM-800, an ERalpha antagonist, or 4) CGS 20267, a nonsteroidal aromatase inhibitor. On PND 14, ewes were hemihysterectomized, and the ipsilateral oviduct and ovary were removed. The remaining uterine horn, oviduct, and ovary were removed on PND 56. Treatment with CGS 20267 decreased plasma E2 levels, whereas EM-800 had no effect compared with CX ewes. Uterine horn weight and length were not affected by EM-800 or CGS 20267 but were decreased in EV ewes on PND 56. On PND 14 and PND 56, treatment with EV decreased endometrial thickness but increased myometrial thickness. The numbers of ductal gland invaginations and endometrial glands were not affected by CGS but were lower in EM-800 ewes on PND 56. Exposure to EV completely inhibited endometrial gland development and induced luminal epithelial hypertrophy but did not alter uterine cell proliferation. Exposure to EV substantially decreased expression of ERalpha, insulin-like growth factor (IGF) I, and IGF-II in the endometrium. Results indicate that circulating E2 does not regulate endometrial gland differentiation or development. Although ERalpha does not regulate initial differentiation of the endometrial glandular epithelium, results indicate that ERalpha does regulate, in part, coiling and branching morphogenesis of endometrial glands in the neonatal ewe. Ablation of endometrial gland genesis by EV indicates that postnatal uterine development is extremely sensitive to the detrimental effects of inappropriate steroid exposure.

Effects of neonatal progestin exposure on female reproductive tract structure and function in the adult ewe.

Endometrial glands are present in all mammalian uteri and produce secretions that are hypothesized to support conceptus (i.e., embryo/fetus and placental membranes) survival and development. In sheep, endometrial gland morphogenesis occurs postnatally and can be epigenetically ablated by chronic neonatal exposure to a progestin from birth, thereby producing an adult uterine gland knock-out (UGKO) phenotype. This study determined the long-term effects of neonatal progestin exposure on adult ovine reproductive tract structure and function. Neonatal ewes were exposed to norgestomet (Nor) from birth to 32 wk of age. Unexposed ewes served as controls. After puberty, adult Nor-treated (n = 6) and control (n = 6) ewes were repeatedly bred at estrus (Day 0) to intact rams of proven fertility. In contrast to a pregnancy rate of 80% for control ewes, pregnancy was never detected on Day 25 after mating (or thereafter) in bred UGKO ewes. Control and Nor-treated ewes were then bred and necropsied on Day 9. Similar numbers of hatched blastocysts were present in uterine flushings from control and Nor-treated ewes. Weights of the ovaries and cervices were not affected by treatment. No histoarchitectural differences between control and Nor-treated ewes were detected for ovaries, oviducts, cervices, or vaginae. However, uterocervical and uterine weight as well as uterine horn length were less for Nor-treated ewes. The uteri of Nor-treated ewes were devoid of endometrial glands and lacked the stromal delineation characteristic of intercaruncular endometrium in control ewes. Endometrial width, area, and lumenal epithelial length were decreased in uteri from Nor-treated ewes, but myometrial width and morphology were not affected. Expression of a number of mRNAs that are expressed predominantly in the endometrial epithelia was not different between uteri from control and from Nor-treated ewes. Collectively, these results indicate that neonatal exposure of ewes to a progestin from birth appears to only affect development of the uterus and not any extrauterine reproductive tract tissues. The infertility of the UGKO ewes appears to result from a lack of endometrial glands and, by extension, of their secretions that are required to support growth and development of peri-implantation conceptuses.

Neonatal ovine uterine development involves alterations in expression of receptors for estrogen, progesterone, and prolactin.

Effects of age on uterine histoarchitecture, cell proliferation, and hormone receptor expression were determined for neonatal ewe lambs from birth (Postnatal Day [PND] 0) to PND 56. Uteri were histologically evaluated and proliferating cell nuclear antigen (PCNA), estrogen receptor alpha (ER-alpha), progesterone receptor (PR), and prolactin receptor (PRL-R) expression were characterized by in situ hybridization (ISH), immunohistochemistry, or both. The most striking feature of neonatal uterine development was the genesis and development of glands in the intercaruncular areas of endometrium. After birth, endometrial glandular epithelium (GE) budded and differentiated into the underlying stroma from the luminal epithelium (LE) between PNDs 1 and 7. Between PNDs 14 and 56, extensive coiling and branching morphogenesis of nascent endometrial glands occurred. By PND 56, the uterine wall appeared to be histoarchitecturally mature. At birth, nuclear PCNA protein was strongly detected in LE. Between PNDs 7 and 56, high levels of PCNA, ER-alpha, and PR gene expression were detected in both nascent and developing GE. Higher levels of PCNA and ER-alpha expression were detected in GE at the tips of developing glands as well as in the surrounding stroma. Progesterone was below detectable limits in serum. Serum estradiol-17beta levels were high on PND 1, increased from PNDs 14 to 28, and declined from PND 42 to PND 56. Serum PRL levels increased from PNDs 1 to 14 and declined thereafter. Using ISH and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, expression of mRNAs for short and long forms of the ovine PRL-R were first detected in nascent GE on PND 7 and increased between PNDs 7 and 56 in proliferating and differentiating GE. These results indicate that 1) uterine gland genesis is initiated between PNDs 1 and 7 after birth and is essentially completed by PND 56; 2) neonatal uterine morphogenesis involves temporal and spatial alterations in cell proliferation and ER-alpha, PR, and PRL-R gene expression; 3) PRL-R expression is a unique marker of GE differentiation and proliferation; and 4) serum estradiol-17beta and PRL levels increase during the onset of GE tubular branching morphogenesis. Results support the hypothesis that neonatal ovine uterine development involves epithelial PRL-R and ER-alpha activation to stimulate and maintain endometrial gland genesis and branching morphogenesis.

Mechanisms regulating norgestomet inhibition of endometrial gland morphogenesis in the neonatal ovine uterus

In many species, endometrial gland adenogenesis occurs neonatally in an ovary- and steroid-independent manner. Chronic exposure of the developing neonatal ovine uterus to norgestomet (NOR) from birth permanently ablates endometrial gland morphogenesis or adenogenesis, creating an adult ovine uterine gland knockout (UGKO) phenotype. This study was conducted to determine the mechanism(s) whereby NOR inhibits adenogenesis in the neonatal ewe. Ewe lambs received no implant or a NOR implant at birth and on postnatal day (PND) 14, and they were necropsied on PND28. Histological analyses of the tracts indicated NOR exposure specifically inhibited endometrial adenogenesis, but no histoarchitectural differences were observed in the oviduct, cervix, or vagina. No effect of NOR treatment was detected on proliferating cell nuclear antigen (PCNA) expression in the endometrial luminal epithelium (LE), stroma, or myometrium. In control (CX) ewes, estrogen receptor α (ER-α) and progesterone receptor (PR) mRNA and protein were expressed strongly in nascent and proliferating glandular epithelium (GE) but were undetected in epithelium of NOR uteri. Expression of c-met and fibroblast growth factor receptor 2IIIb (FGFR2IIIb) mRNA was detected in the LE and GE of CX uteri. In NOR uteri, c-met was expressed in the LE similar to CX uteri, but FGFR2IIIb mRNA levels were lower than in the LE of CX uteri. Uterine hepatocyte growth factor (HGF), the ligand for c-met, and FGFR2IIIb mRNA expression was substantially lower in NOR ewes, but expression of FGF-7 and FGF-10 mRNAs, ligands for FGFR2IIIb, was unaffected. These results indicate that NOR disrupts endometrial adenogenesis by ablating epithelial ER-α expression and altering expression of paracrine growth factors and/or receptors involved in epitheliomesenchymal interactions. Likewise, these mechanisms are proposed to be important regulators of normal uterine gland morphogenesis in the neonate. Mol. Reprod. Dev. 57:67–78, 2000. © 2000 Wiley-Liss, Inc.

Mechanisms regulating norgestomet inhibition of endometrial gland morphogenesis in the neonatal ovine uterus.

In many species, endometrial gland adenogenesis occurs neonatally in an ovary- and steroid-independent manner. Chronic exposure of the developing neonatal ovine uterus to norgestomet (NOR) from birth permanently ablates endometrial gland morphogenesis or adenogenesis, creating an adult ovine uterine gland knockout (UGKO) phenotype. This study was conducted to determine the mechanism(s) whereby NOR inhibits adenogenesis in the neonatal ewe. Ewe lambs received no implant or a NOR implant at birth and on postnatal day (PND) 14, and they were necropsied on PND28. Histological analyses of the tracts indicated NOR exposure specifically inhibited endometrial adenogenesis, but no histoarchitectural differences were observed in the oviduct, cervix, or vagina. No effect of NOR treatment was detected on proliferating cell nuclear antigen (PCNA) expression in the endometrial luminal epithelium (LE), stroma, or myometrium. In control (CX) ewes, estrogen receptor alpha (ER-alpha) and progesterone receptor (PR) mRNA and protein were expressed strongly in nascent and proliferating glandular epithelium (GE) but were undetected in epithelium of NOR uteri. Expression of c-met and fibroblast growth factor receptor 2IIIb (FGFR2IIIb) mRNA was detected in the LE and GE of CX uteri. In NOR uteri, c-met was expressed in the LE similar to CX uteri, but FGFR2IIIb mRNA levels were lower than in the LE of CX uteri. Uterine hepatocyte growth factor (HGF), the ligand for c-met, and FGFR2IIIb mRNA expression was substantially lower in NOR ewes, but expression of FGF-7 and FGF-10 mRNAs, ligands for FGFR2IIIb, was unaffected. These results indicate that NOR disrupts endometrial adenogenesis by ablating epithelial ER-alpha expression and altering expression of paracrine growth factors and/or receptors involved in epitheliomesenchymal interactions. Likewise, these mechanisms are proposed to be important regulators of normal uterine gland morphogenesis in the neonate.

Production and lambing rate of blastocysts derived from in vitro matured oocytes after gonadotropin treatment of prepubertal ewes.

The aim of this study was to evaluate the effect of gonadotropin treatment on the in vitro maturation, blastocyst production, and developmental potential to term of oocytes collected from Sardinian neonatal and prepubertal ewes at 4 to 6 wk of age. Cumulus-oocyte complexes were recovered at 24 h after withdrawal of a 1/6th size progestagenated pessary from the donors, of which each received 120 IU FSH/LH and 400 IU PMSG in a single dose 36 h before sponge removal. Treated donors produced a greater (P<.01) number of oocytes per animal (86.2 +/-7.9) compared with slaughterhouse (untreated) prepubertal ewes (55.5+/-6.1) of the same age or with treated neonatal ewes (6.1+/-0.7) 10 d old. During oocyte maturation, there were no differences in the percentage of germinal vesicle break-down (78.08 vs. 74.24), metaphase I (89.13 vs. 87.18), and metaphase II (77.91 vs. 76.38) when evaluated after 8, 14, and 24 h of maturation, respectively, between oocytes from treated and slaughterhouse (untreated) prepubertal ewes. The embryo cleavage (71.1 vs. 73.7) and blastocyst rates (22.2 vs. 19.8) were similar in the treated and the untreated prepubertal ewes after transfer of in vitro matured oocytes into ligated oviducts of temporary recipients. The in vitro viability rates of vitrified blastocysts (81.2 vs. 76.9) and the in vivo survival rates (46.1 vs. 50.0) of embryos derived from in vitro matured and in vivo fertilized oocytes showed no difference. The data suggest that gonadotropin treatment increases oocyte production per animal but has no effect on oocyte quality because embryo production and lambing rates of blastocysts derived from in vitro matured oocytes were not markedly different from those derived from untreated prepubertal ewes of the same age.

Gonadotrophin levels and ovarian development in the neonatal ewe lamb.

Ovarian weight has been shown to increase markedly in the newborn ewe lamb, and factors which might contribute to this growth were examined. Follicle development was studied in the ovaries of 28 lambs aged 0, 2, 4, 6, 8 and 10 weeks. Plasma samples, pituitaries and one ovary were assayed for gonadotrophin or steroid hormone content. No significant differences between age groups were found in pituitary LH concentration or mean plasma LH. Pituitary FSH concentration increased with age, except for a small decrease at 8 weeks, but no significant changes were seen in plasma FSH. Ovarian progesterone and oestradiol concentrations did not appear to be associated with follicular development. Growing follicles were most numerous at 2 weeks whilst total vesicular follicles reached a peak at 4 weeks. Most vesicular follicles were less than 1.4 mm in diameter. Advanced atresia in vesicular follicles became apparent by 4 weeks although early signs were present in younger lambs. Fluctuations in gonadotrophin levels do not appear to be responsible for variations in number and size of vesicular and growing follicles in the lamb ovary. Other possible explanations are discussed.