Fertilization and early embryology: The role of calcium in mammalian oocyte maturation and egg activation

The maturation of the immature oocyte and the fertilizationof a mature egg are two absolute prerequisites for mammalianembryo development. There is increasing evidence in mammalsthat both oocyte maturation and egg activation at fertilizationare controlled by changes in intracellular free Ca²⁺ levels.The role of Ca²⁺ changes at fertilization is clear in that theyare both required and sufficient for egg activation. However,it is not established how the sperm causes Ca²⁺ changes in eggsat fertilization, nor how different patterns of Ca²⁺ changeaffect embryo development. The role of Ca²⁺ in triggering oocytematuration is less clear, although preventing intracellularCa²⁺ changes can inhibit meiotic maturation at specific stages.Studies on how Ca²⁺ regulates meiosis and fertilization in mammalsmay provide new insights into the causes of failed fertilizationin human IVF procedures.     

Oxygen concentration during mouse oocyte in vitro maturation affects embryo and fetal development

BACKGROUND: Little is known of how the oxygen environment in the ovarian follicle affects oocyte and embryo development, but this has an important impact on the conditions used for in vitro maturation (IVM) of oocytes. We investigated the effect of varying oxygen concentrations during IVM on subsequent pre and post-implantation development. METHODS: IVM of mouse cumulus-oocyte complexes (COCs) was performed under 2, 5, 10 or 20% O(2) (6% CO(2), balance N(2)). In vivo-matured COCs were collected post ovulation. Embryos were generated by IVF and culture. Blastocyst development, cell number and apoptosis were assessed, and fetal and placental outcomes analysed following embryo transfer at day 18 of pregnancy. RESULTS: Oxygen concentration during IVM did not affect oocyte maturation or subsequent fertilization, cleavage and blastocyst development rates. Maturation of oocytes under 2% O(2) increased blastocyst trophectoderm cell number compared with all groups and numbers at 5% were higher than 20% (both P < 0.05). Percentage of apoptotic cells was increased in blastocysts developed from 2% O(2)-matured oocytes, compared with maturation at 5% O(2) or in vivo (P < 0.05). Rates of embryo implantation and development into a viable fetus were not altered by IVM oxygen. However, fetal weight was reduced following oocyte maturation at 5% O(2) compared wiht 20% O(2) and maturation at 5% O(2) also reduced placental weight, when compared with in vivo-matured oocytes (both P < 0.05). CONCLUSIONS: Level of O(2) exposure during oocyte maturation can alter the cellular composition of blastocysts, but these changes in cell number do not correlate with the altered fetal and placental outcomes after transfer.     

Rescue of oocytes from antral follicles of cryopreserved mouse ovaries: competence to undergo maturation, embryogenesis, and development to term

Only primordial and primary follicles of frozen-thawed mouse ovaries survive after grafting to the ovarian bursa; large secondary follicles and antral follicles together with the oocytes contained in them degenerate. This study was undertaken to determine whether fully grown oocytes isolated from the antral follicles of frozen-thawed mouse ovaries are viable and can be rescued to undergo maturation, fertilization, and embryo development in vitro. Ovaries were cryopreserved after removal from 22-day-old (C57BL/6J x SJL/J)F(1) mice, with or without prior priming with equine chorionic gonadotrophin, and fresh non-frozen ovaries were used as controls. Only cumulus cell-denuded oocytes were recovered from frozen unprimed ovaries while both cumulus cell-enclosed and denuded oocytes were retrieved from frozen primed ovaries. Oocytes from both groups of frozen-thawed ovaries were able to undergo maturation, fertilization, and development to the blastocyst stage in vitro, though at lower percentages than oocytes from control unfrozen ovaries. Moreover, 19% of 2-cell stage embryos derived from frozen-thawed primed ovaries, compared with 42% of embryos derived from control primed ovaries, developed to term after transfer to pseudopregnant foster mothers (not significantly different). Therefore, fully grown oocytes in antral follicles survive the cryopreservation protocol, as demonstrated by maturation, fertilization and embryo development in vitro, and development to term after embryo transfer.     

Oocyte activation and Ca(2+) oscillation-inducing abilities of mouse round/elongated spermatids and the developmental capacities of embryos from spermatid injection

To investigate differences in fertilization mechanisms and the potential clinical use of round/elongated spermatid, we conducted detailed studies of oocyte activation and Ca(2+) oscillation-inducing abilities in these immature sperm cells and compared these abilities against those of mature spermatozoa. When round spermatids from B(6)D(2)F(1) mice were injected, none of the oocytes was activated and no intracellular Ca(2+) ([Ca(2+)](i)) increases were observed. Elongated spermatids could induce activation normally in 87% of injected oocytes, but Ca(2+) oscillation could not be induced at all and most of the oocytes (94%) exhibited only several transient [Ca(2+)](i) rises (transient patterns). Because normal offspring could be obtained when embryos through elongated spermatid injection were transferred to foster mothers, it seems that a normal oscillation pattern of [Ca(2+)](i) is not essential for normal fertilization and embryo development. [Ca(2+)](i) patterns of injected oocytes changed from transient patterns to oscillation patterns while the injected immature sperm cells were maturing to spermatozoa. Dissociations were seen between the timing of appearance of oocyte activation and that of Ca(2+) oscillation-inducing abilities in maturing sperm cells. These dissociations may be due to differences in the thresholds to oocyte activation and Ca(2+) oscillation-inducing factor for inducing oocyte activation and Ca(2+) oscillation.     

C-ERBB2促小鼠卵母细胞成熟及在表皮生长因子调控小鼠卵母细胞体外成熟中的作用

目的探讨C-ERBB2对小鼠卵母细胞成熟的影响及对表皮生长因子在卵母细胞成熟调控中的作用。方法采用卵母细胞体外培养法,研究反义C-ERBB2寡脱氧核苷酸(C-ERBB2ASODN)对卵母细胞体外自发成熟及对表皮生长因子(EGF)促卵母细胞成熟的影响;利用RT-PCR检测卵母细胞中C-ERBB2mRNA的表达及在不同处理因素作用下卵母细胞成熟过程中C-ERBB2mRNA表达的变化。结果C-ERBB2ASODN能呈剂量依赖方式抑制卵母细胞的生发泡破裂(GVBD)率和第一极体(PBⅠ)排出率,并且能抑制EGF对卵母细胞的促成熟作用。卵母细胞中存在C-ERBB2mRNA,EGF可以增强其表达,C-ERBB2ASODN可减弱EGF促表达的作用。结论C-ERBB2与卵母细胞的成熟密切相关。EGF可直接促进裸卵的成熟分裂,其机制可能与上调卵母细胞C-ERBB2的表达有关。     

Effects of epidermal growth factor in the final stages of nuclear and cytoplasmic oocyte maturation in humans

Evidence has accumulated in mammals suggesting a positive rolefor epidermal growth factor (EGF) as an inducer of oocyte maturation.The potential use of EGF as inducer of cytoplasmic and nuclearmaturation was tested in women with > 10 oocytes retrievedin in-vitro fertilization (IVF), since we have previously observedthat such oocytes are immature. Oocytes from 17 high responderswere randomly allocated to one of the three treatment groupsupon retrieval: control receiving no EGF (n = 93), 1.0 ng/mlEGF (n = 92) and 10.0 ng/ml EGF (n = 77) for 6 h before insemination.The rates of fertilization were respectively 54.6, 59.0, and46.1%, suggesting that EGF is not effective at this maturationalstage after this length of exposure. Embryo development wasfurther analysed by the appearance of the embryos under thedissecting microscope and the number of blastomeres developed48 h after insemination. No difference between groups was observedconsidering the number of blastomeres developed. However, embryosderived from oocytes treated with 10 ng/ml EGF displayed a worseappearance under the microscope. It is concluded that a 6 hincubation with EGF does not seem to affect cytoplasmic maturationin oocytes obtained after gonadotrophin treatment, as ascertainedby the rate of fertilization following oocyte insemination.     

Meiotic spindle configuration is differentially influenced by FSH and epidermal growth factor during in vitro maturation of mouse oocytes

BACKGROUND: To ascertain whether different hormonal treatment protocols could affect metaphase II (MII) spindle morphology, meiotic spindle organization was detected in prepubertal mouse oocytes matured under conditions allowing spontaneous, FSH- or epidermal growth factor (EGF)-dependent meiotic maturation. METHODS: Oocyte-cumulus complexes (OCCs) were matured either spontaneously (control; n=270) or in the presence of hypoxanthine (Hx) plus FSH (n=400) or EGF (n=370). Spindles were detected by immunofluorescence analysis. In vivo ovulated (IVO) oocytes were processed similarly. RESULTS: IVO oocytes displayed spindles underlying the oolemma and with focused poles marked by spots of gamma-tubulin, whereas the majority (89%) of control oocytes had barrel-shaped spindles, positioned away from the oolemma, and with gamma-tubulin distributed along microtubules. Similar configuration/localization was found in 85% of the oocytes matured in vitro in the presence of Hx and FSH. In the presence of Hx-EGF, 35% of the oocytes showed spindles with an IVO-like configuration, although gamma-tubulin was homogeneously distributed throughout microtubules. Independently of spindle shape, 52% of EGF-stimulated oocytes had spindles positioned near the oolemma, in comparison to just 24% of FSH-treated and 13% of control oocytes. CONCLUSIONS: These results indicate that FSH and EGF can differently affect meiotic spindle morphology, and that EGF might be a stronger contributor than FSH to the acquisition of oocyte competence.     

Analysis of the human sperm centrosomal function and the oocyte activation ability in a case of globozoospermia,by ICSI into bovine oocytes

BACKGROUND: Centrosomal function and oocyte activation ability of human sperm from a case of globozoospermia was assayed by heterologous ICSI into bovine oocytes. METHODS: Microtubules and chromatin configuration in bovine oocytes were examined by immunofluorescence after heterologous ICSI with human sperm from two fertile donors and from a globozoospermic man. RESULTS: The microtubule array from the sperm centrosome, the 'sperm aster' and the male pronucleus were observed in bovine oocytes, following ICSI with round-headed sperm from a globozoospermic man. The rate of sperm aster formation and the rate of male pronuclear formation in the bovine oocytes injected with fertile donor sperm were 57.9 and 92.5% respectively; the respective values for oocytes injected with round-headed sperm without artificial oocyte activation were 15.8 and 31.0%. Ethanol activation after ICSI improved male pronuclear formation (84.9%) but not sperm aster formation rate (32.3%) of the globozoospermic patient. CONCLUSIONS: These data indicated that sperm from this patient with globozoospermia have centrosomal dysfunction and low ability for oocyte activation compared with fertile donor sperm. The centrosomal dysfunction may be one of the reasons for infertility in this patient.     

Influence of blood clots in the cumulus complex on oocyte fertilization and cleavage

Since multiple ovarian punctures are performed during oocyteretrieval, the likelihood of blood contaminating the follicularfluid is high. The purpose of this study was to determine whetherthe presence of blood clots, which are frequently seen in thecumulus of oocytes retrieved under ultrasound guidance, hasany effect on fertilization and subsequent embryo cleavage andto identify oocyte characteristics which may predict these events.Oocytes were morphologically graded and the presence of bloodclots in the cumulus was recorded. Cases in which the male partnerhad a total motile sperm count < 20 ? 10⁶ on the day of inseminationwere excluded from the logistic regression analysis. The presenceof blood clots in the cumulus was a negative predictor of cleavage[odds ratio (OR) = 0.54]. The results indicate that an oocytewith optimal quality is one which is spherical (OR = 5.45),has an expanded corona (OR = 3.80) and has no blood clots inthe cumulus complex.     

The concentration of inhibin B in follicular fluid: relation to oocyte maturation and embryo development

BACKGROUND: The objective of the present study was to investigate the correlation between inhibin B and estradiol levels in follicular fluid (FF) with the quality of subsequent embryo development from in-vitro fertilized oocytes aspirated from the same follicle. METHODS: A total of 156 infertile women undergoing controlled ovarian stimulation for IVF and embryo transfer was recruited to the present study. Prospectively, 233 FF samples and matched mature oocytes were studied. Concentrations of inhibin B and estradiol were determined by enzyme-linked immunosorbent assay (ELISA) and immunofluorometric assay (IFMA) respectively. RESULTS: Inhibin B levels in FF were significantly correlated with embryo scores on days 2 and 3 (48 and 72 h after oocyte retrieval). In contrast, both inhibin B and estradiol levels in FF were inversely related to age. Furthermore, FF inhibin B levels were inversely associated with serum FSH levels on day 3 of the menstrual cycle, which was believed to reflect the ovarian reserve. CONCLUSION: Inhibin B in FF may serve as an effective marker of follicular development and a useful predictor of quality of embryo. In addition, quality of oocyte is age-related and declines as age increases.     

Birth of a healthy infant after in vitro oocyte maturation and ICSI in a woman with diminished ovarian response: case report

In vitro maturation of oocytes (IVM) has been developed as a treatment option for subjects with good prognosis in assisted reproduction. We present successful IVM treatment in connection with a woman from whom low numbers of embryos were obtained after repeated failed conventional IVF cycles. A 35 year old woman, after 5 years infertility and two intrauterine insemination and three conventional IVF cycles, underwent first an IVM cycle with low dose FSH stimulation, and after failure, another natural IVM cycle. Three oocytes were obtained. After 36 h of IVM the oocytes had reached metaphase II stage, and fertilization using ICSI resulted in one 4-cell stage embryo, which was transferred 2 days later. The result was an uneventful pregnancy and birth of a healthy female infant weighing 4150 g. IVM may be an option for women from whom only low numbers of oocytes are obtained after gonadotrophin stimulation.     

Comparison of Ca2+ and CaMKII responses in IVF and ICSI in the mouse

Novel methods of egg activation in human assisted reproductive technologies and animal somatic cell nuclear transfer are likely to alter the signalling process that occurs during normal fertilization. Intracytoplasmic sperm injection (ICSI) bypasses the normal processes of the acrosome reaction, sperm-egg fusion, and processing of the sperm plasma membrane, as well as alters some parameters of intracellular calcium ([Ca(2+)](i)) dynamics (reported previously by Kurokawa and Fissore (2003)). Herein, we extend these studies to determine if ICSI alters the activity of the Ca(2+)-dependent protein, Ca(2+)/calmodulin-dependent kinase II (CaMKII), which is responsible for the completion of meiosis in vertebrate eggs. After ICSI or in vitro fertilization (IVF), individual mouse eggs were monitored for their relative changes in both [Ca(2+)](i) and CaMKII activity during the first [Ca(2+)](i) rise and a subsequent rise associated with second polar body extrusion. The duration of the first [Ca(2+)](i) rise was greater in ICSI than in IVF, but the amplitude of the rise was transiently higher for IVF than ICSI. However, a similar mean CaMKII activity was observed in both procedures. During polar body extrusion, the amplitude and duration of the Ca(2+) rises were increased by a small amount in ICSI compared with IVF, whereas the CaMKII activities were similar. Thus, compared with IVF, ICSI is not associated with decreased or delayed CaMKII activity in response to these Ca(2+) signals in the mouse.     

Role of protein tyrosine phosphorylation in the thapsigargin-induced intracellular Ca(2+) store depletion during human sperm acrosome reaction

During human sperm capacitation, an increase in phosphotyrosine content of specific proteins results partially from an increase in the intracellular free Ca(2+) concentrations. In the present study, the inter-regulation between protein phosphotyrosine content and the intracellular Ca(2+) concentration during the thapsigargin treatment of capacitated human sperm was investigated. The involvement of a tyrosine kinase pathway in the thapsigargin-induced acrosome reaction was also investigated. In response to thapsigargin, two sperm subpopulations, called LR (low responsive) and HR (high responsive), according to their increase in intracellular Ca(2+), were observed. In addition to their high increase in intracellular Ca(2+), sperm from the HR population expressed a higher protein phosphotyrosine content, and a higher proportion (P < 0.05) of them underwent the acrosome reaction in response to thapsigargin, as compared with LR sperm. Although the tyrosine kinase inhibitor PP2 abolished the thapsigargin-induced increase in protein phosphotyrosine content, it did not affect the intracellular Ca( 2+) concentration or the percentage of acrosome-reacted sperm. The inability of an src-related tyrosine kinase inhibitor to block the thapsigargin-mediated Ca(2+) increase and acrosomal exocytosis suggests that, during the acrosome reaction, the signalling pathway mediated by src-related tyrosine kinases is involved upstream of the capacitative Ca(2+) entry.     

Ovary and ovulation: Application of image analysis cytometry in fofficular fluid cells obtained from in-vitro fertilization cycles: relationships to patient's age, oocyte maturity, fertilizability and in-vitro fertilization outcome

In an in-vitro fertilization (LVF)/embryo transfer pro grammegranulosa cells obtained from 59 individual preovulatory follicleswere analysed using multiparameter image analysis cytometry,in an attempt to determine whether their morphometric and DNA-cytometricparameters could prove useful in assessing follicle and oncytematurity and in predicting fertilizabifity and outcome of theseIVF cycles. Almost all morphometric and DNA- cytometric parameterswere not correlated with either the patient's age or oocytematurity, and did not predict oocyte fertilization or occurrenceof a clinical pregnancy. The only possible relevant parameterwhich, despite its inverse correlation to total luteinizinghormone administration, also proved to be inversely correlatedto pregnancy outcome (in the seven cases in which a pregnancyoccurred), was the percentage of granulosa cell nuclei withincreased DNA content (>5c). Finally, if granulosa cellsdo not reveal euploid polyploidization in spontaneous or inducedovulatory cycles, the detected cells with increased DNA contentshould be interpreted as aneuploid, i.e. with chromosomal aberrations,and so their presence could also be discussed in connectionwith the hypothetical risk of prospective neoplastic transformationof the tissue.     

Expression of PTHrP and PTHR (PTH/PTHrP-r) mRNAs and polypeptides in bovine ovary and stimulation of bovine blastocyst development in vitro following PTHrP treatment during oocyte maturation

Parathyroid hormone related protein (PTHrP) and its receptor have well-established roles in the development and regulation of many tissues, including bone and mammary gland. The objectives of this study were: (1) to characterize the distribution of mRNAs encoding parathyroid hormone (PTH)-related protein (PTHrP) and receptor (PTHR) in bovine ovary; (2) to characterize the distribution of PTHrP and PTHR polypeptides in bovine ovary; (3) to examine the influences of PTHrP (1–141) treatment during bovine oocyte maturation in vitro on blastocyst development. mRNAs encoding PTHrP and PTHR were detected by in situ hybridization methods in oocytes, and granulosa cells in all follicles from primordial to large antral. PTHrP and PTHR polypeptides displayed distinct distribution patterns with PTHrP polypeptides primarily confined to oocytes from primordial to large antral follicles. PTHrP polypeptides were detectable but at a reduced level in ovarian stroma and in granulosa and thecal layers. PTHR polypeptides were detected in oocytes of all follicular stages but were predominantly found in ovarian stroma, granulosa and theca follicular layers. Supplementation of serum-free cSOFMaa oocyte maturation medium with PTHrP (1–141) resulted in a concentration-dependent increase in development to the blastocyst stage in vitro. The results suggest that granulosa cells may be a primary site of PTHrP production and release. Oocytes from all follicular stages stained strongly for PTHrP polypeptides and PTHrP enhanced development to the blastocyst stage in vitro. Accepted: 12 October 2000     

Fertilization promoting peptide, a tripeptide similar to thyrotrophin-releasing hormone, stimulates the capacitation and fertilizing ability of human spermatozoa in vitro

Recent studies have demonstrated that a prostatic tri-peptidesimilar in structure to thyrotrophin-releasing hormone (TRH)can stimulated the in-vitro capacitation and fertilizing abilityof epididymal mouse spermatozoa. Therefore we have proposedthat this tripeptide be referred to as fertilization promotingpeptide (FPP). Using chlortetracycline fluorescence analysisand the hamster oocyte penetration test (HOPT), we have obtainedevidence that FPP can also promote the capacitation and fertilizingability of ejaculated human spermatozoa in vitro. FPP (25–200nM) caused a significant increase in the proportion of B-patterncapacitated cells and a decrease in the proportion of F-patternuncapacitated cells, with no significant stimulation of acrosomalexocytosis. Comparison of FPP with two structurally similartripeptides, TRH and pyro-glutamyl phenylalanylprolineamide,at 50 nM revealed that only FPP could significantly promotecapacitation. Finally, after a brief exposure to progesteroneto induce acrosomal exocytosis in capacitated cells, FPP-treatedsuspensions penetrated a significantly higher proportion ofoocytes than the untreated controls when assessed in the HOPT.The presence of FPP in human seminal plasma at concentrationssimilar to those used here suggests that, in vivo, FPP may playa positive role in promoting human sperm function.     

Maturation of human spermatozoa (from selected epididymides of prostatic carcinoma patients) with respect to their morphology and ability to undergo the acrosome reaction

Spermatozoa were recovered form three regions of the epididymisof six prostatic carcinoma patients. After washing and incubatingfor 3 h in Ham's F-10 medium, with or without 5 µM A23187for the last 30 min, spermatozoa were tested for vitality byhypotonic swelling and permeated with methanol to detect theacrosome with peanut agglutinin. Whereas the extent of spontaneousacrosome reactions was similar for spermatozoa from all regionsof the duct, 17 and 28% of spermatozoa from all regions of theduct, 17 and 28% of spermatozoa from the corpus and cauda epididymidisrespectively, responded to stimulation by A23187 with acrosomereactions but there was no stimulation by A23187 of spermatozoafrom the efferent ducts. The percentage of morphologically normalspermatozoa increased stepwise towards the distal regions, withabnormalities being mostly enlarged heads in more proximal regions:they were largely absent form the cauda epididymidis. Spermhead swelling was similarly observed in cynomolgus monkey spermatozoafrom the caput epididymidis but not the more distal regions.These forms were not observed when spermatozoa were fixed beforesmearing, indicating that they were artefacts of sperm preparation.The changes in the susceptibility of non-fixed epididymal spermatozoato produce morphological artefacts and the gain in their acrosomalresponse to ionophore demonstrate maturational changes of spermatozoain the human epididymis.     

Infertility: A randomized, assessor-blind, group-comparative efficacy study to compare the effects of Normegon(R) and Metrodin(R) in infertile female patients undergoing in-vitro fertilization

A randomized, assessor-blind, group-comparative study was performedto compare the efficacy of Normegon^® [75 IU follicle stimulatinghormone (FSH) and 25 IU luteinizing hormone (LH)] and Metrodin^®(75 IU FSH and <1.25 IU LH) in infertile women undergoingin-vitro fertilization (IVF) and embryo transfer. None of thepatients were pituitary-suppressed by means of gonadotrophin-releasinghormone (GnRH)-agonist treatment. They were randomized in blocksof five with a ratio between treatment with Normegon and withMetrodin of 3: 2. A total of 158 patients started hormonal treatment,i.e. 93 patients with Normegon and 65 patients with Metrodinand a total of 248 cycles were performed. Evaluation of firsttreatment cycles included statistical analysis of the totalnumber of ampoules, number of follicles (14 mm), serum oestradiolconcentrations on the day of HCG (10 000 IU) administration,the number of oocytes retrieved and the ongoing pregnancy rateper attempt and per transfer. For none of these parameters weresignificant differences revealed. In both groups the medianduration of stimulation was 7 days and the median number ofampoules used was 21. Overall, the duration of treatment wasshort in order to prevent as much as possible endogenous LHrises. The overall ongoing pregnancy rate per transfer of allcycles was 21% in the Normegon group and 19% in the Metrodingroup. Analysis of completed treatment cycles (n = 90) withpremature rises of LH >10.0 IU/l and/or progesterone >1.0ng/l revealed a relatively high incidence (23%) of fertilizationfailure and poor embryo quality, but the ongoing pregnancy rateper transfer was still 22%. These data suggest that prematurerises of LH and progesterone are deleterious for oocyte qualitybut may not affect the endocrine environment of the endometrium.In conclusion, Normegon is an efficacious preparation for theinduction of ovarian stimulation in infertile women undergoingIVF.     

Ovary: Immunolocalization of transforming growth factor-{beta}1 and transforming growth factor-{beta}2 in the mouse ovary during gonadotrophin-induced follicular maturation

An immunohistochemical approach was utilized to evaluate thecellular distribution of transforming growth factor-₁ (TGF₁)and transforming growth factor ₂ (TGF₂) at different stagesof follicle development in the prepubertal mouse ovary underthe following conditions: (i) after pregnant mare's serum gonadotrophin(PMSG) treatment; (ii) after PMSG and human chorionic gonadotrophin(HCG) treatment; (iii) after PMSG and HCG treatment plus mating.In the immature ovary, TGFF₁ and TGF₂ immunoreactivities arelocalized in theca and granulosa cells and in oocytes. AfterPMSG treatment, TGF₁ and TGF₂ immunoreactivities are localizedin granulosa cells; in addition, TGF₂ staining is noted in thematrix surrounding antral cells. Staining for both TGF₁ andTGF₂ drops in the theca but persists in the oocyte. PMSG plusHCG treatment results in a significant increase in TGF₁ andTGF₂ immunoreactivity in the theca and in the maintenance ofTGF₁ staining in both basal granulosa cells and cumulus cellswhereas TGF₂ immunoreactivity is essentially localized in thematrix surrounding cumulus cells. Staining for TGF₁ and TGF₂persists in the oocyte. Following PMSG plus HCG treatment andmating, TGF₁ immunoreactivity is localized in the luteal cellsof corpora lutea and TGF₂ shows a similar localization pattern.This study provides evidence that TGF₁ and TGF₂ peptides areexpressed in specific cell types during induced follicular maturationin the mouse ovary.