Genetic and phenotypic influences on copulatory plug survival in mice.

Across a diversity of animals, male seminal fluid coagulates upon ejaculation to form a hardened structure known as a copulatory plug. Previous studies suggest that copulatory plugs evolved as a mechanism for males to impede remating by females, but detailed investigations into the time course over which plugs survive in the female's reproductive tract are lacking. Here, we cross males from eight inbred strains to females from two inbred strains of house mice (Mus musculus domesticus). Plug survival was significantly affected by male genotype. Against intuition, plug survival time was negatively correlated with plug size: long-lasting plugs were small and relatively more susceptible to proteolysis. Plug size was associated with divergence in major protein composition of seminal vesicle fluid, suggesting that changes in gene expression may play an important role in plug dynamics. In contrast, we found no correlation to genetic variation in the protein-coding regions of five genes thought to be important in copulatory plug formation (Tgm4, Svs1, Svs2, Svs4 and Svs5). Our study demonstrates a complex relationship between copulatory plug characteristics and survival. We discuss several models to explain unexpected variation in plug phenotypes.

Effect of A23187 or angiotensin II on ovarian metalloproteinase inhibitors and steroidogenesis in rats.

The present study examined the effect of the calcium ionophore A23187 or angiotensin II (AII) on the expression of ovarian metalloproteinase inhibitor and activity in rat granulosa cells and intact ovaries. Granulosa cells were collected from rats primed with pregnant mares' serum gonadotrophin (PMSG) and cultured for 24 h with A23187, AII, or the AII receptor antagonist, saralasin, in the presence or absence of LH. Metalloproteinase inhibitor activity and progesterone concentrations were determined in the media. In the A23187 experiment, addition of A23187 to granulosa cells, cultured without LH, decreased inhibitor activity, especially at the concentrations of 10 and 100 mumol l-1 (decrease to 33 +/- 7% and 31 +/- 5% of control culture values, respectively). Addition of LH to the media increased inhibitor activity 3.04 +/- 0.39 times compared with the control; however, A23187 (10 and 100 mumol l-1), in the presence of LH, decreased inhibitor activity by approximately 67%. The ionophore had disparate effects on progesterone production. Without LH, A23187 increased progesterone production by 2.96 +/- 0.47 times at 10 mumol l-1 and by 5.53 +/- 0.65 times at 100 mumol l-1. However, in LH-stimulated cells, progesterone was inhibited by A23187 at 1 and 10 mumol l-1 but was unchanged at 100 mumol l-1. In the angiotensin experiment, addition of AII (0-10,000 nmol l-1) or saralasin (1 mumol l-1) did not affect inhibitor activity or progesterone concentrations compared with control values in the absence or presence of LH. For the angiotensin experiment in vivo, PMSG-primed rats were injected with hCG followed by saralasin (10 mmol l-1) 1 or 3 h later and killed at 4, 8, or 12 h after hCG. Expression of the ovarian tissue inhibitor of metalloproteinase-1 (TIMP-1) increased by 1.7 times at 4 h, 3.3 times at 8 h, and 3.0 times at 12 h after hCG compared with values in ovaries collected at the time of hCG injection. Administration of saralasin at 1 or 3 h after hCG had no effect on expression of TIMP-1 or on serum concentrations of progesterone or oestradiol. In summary, A23187 decreased granulosa cell-derived inhibitor activity, whereas All had no effect. We propose that calcium may play a role in modulating proteolysis associated with ovulation, while AII does not appear to regulate ovarian metalloproteinase inhibitor activity.

Regulation of granulosa cell-derived ovarian metalloproteinase inhibitor(s) by prolactin.

Increased prolactin concentrations are known to inhibit the ovarian proteolytic enzyme cascade associated with follicular rupture. It is not known whether there is also an effect of prolactin on endogenous proteinase inhibitors such as the tissue inhibitors of metalloproteinases (TIMPs). We sought to study the effect of prolactin on ovarian metalloproteinase inhibitors in cultured rat granulosa cells. Granulosa cells were cultured for 24 h with prolactin (0-1000 ng ml-1) in the absence or presence of LH. Metalloproteinase inhibitor activity in the conditioned culture media was measured by a colorimetric assay. Prolactin at 1000 ng ml-1 increased inhibitor activity by 2.86 +/- 0.63 times. Expression of mRNA encoding TIMP-1 measured by Northern analysis increased by 2.34 +/- 0.34 times with 100 ng prolactin ml-1 and by 2.43 +/- 0.42 times with 1000 ng prolactin ml-1 compared with control cultures (no LH, no prolactin). In the presence of LH, expression of mRNA encoding TIMP-1 and inhibitor activity increased by 2.60 +/- 0.6 and 4.60 +/- 0.54 times, respectively. However, no further change in mRNA expression or inhibitor activity was apparent with the addition of prolactin to LH-treated cultures. Prolactin had no effect on expression of mRNA encoding TIMP-3 in the absence or presence of LH, although LH stimulated a 1.7-fold increase in mRNA encoding TIMP-3 compared with controls. Addition of prolactin had no effect on media concentrations of oestradiol or progesterone. These data demonstrate that metalloproteinase inhibitor activity increases with increasing doses of prolactin; however, when LH was added, this effect was no longer seen. With an increase in metalloproteinase inhibitor activity, tissue metalloproteinase action could be decreased, providing a possible explanation for the local inhibition on pre- and peri-ovulatory pathways by hyperprolactinaemia.

Collagenase, gelatinase, and proteoglycanase messenger ribonucleic acid expression and activity during luteal development, maintenance, and regression in the pseudopregnant rat ovary.

In the present study, we examined the roles of the matrix metalloproteinases collagenase, 72-kDa and 92-kDa gelatinase, and proteoglycanase in the tissue remodeling that occurs during luteal development and regression, using a pseudopregnant rat model. Pseudopregnancy was induced in immature female rats by eCG/hCG priming, and animals (n = 3 to 4 per time point) were killed on Days 1, 2, 4, 8, 12, 14, or 16 of pseudopregnancy (Day 0 = time of hCG administration). Ovaries were then removed and analyzed for either matrix metalloproteinase mRNA expression or activity. During luteal development (Day 1 of pseudopregnancy), activity of both collagenase (p = 0.009) and gelatinase (p = 0.0003), but not proteoglycanase (p > or = 0.05), was significantly greater than at all other time points. In accord with gelatinase activity, transcript levels of this enzyme were elevated at Day 1 of pseudopregnancy. Specifically, gelatinase transcript levels for the 72-kDa and 92-kDa enzymes were greatest at Day 1 (p = 0.0003 and p = 0.001, respectively), decreased 3-fold by Day 2,4-fold by Day 4, and reached 10-fold lower levels by Days 8 and 12 of pseudopregnancy. Proteoglycanase transcript could not be detected by Northern analysis during luteal development or any other time point examined in the current study. During the period of luteal maintenance (approximately Days 4-8 of pseudopregnancy in the rat), collagenase and gelatinase displayed basal levels of activity, but only proteoglycanase activity was elevated compared to luteal development levels of this enzyme. During luteal regression (Days 12-14 of pseudopregnancy in the rat), all enzymes displayed basal levels of enzyme activity. In accord with gelatinolytic activity during luteal regression, both 72-kDa and 92-kDa gelatinase mRNA were detectable at baseline levels. In contrast to the baseline levels of collagenolytic activity during luteal regression, collagenase transcript displayed peak values (approximately 8-fold greater than Day 1 levels; p = 0.004) at Day 12 of pseudopregnancy. It is concluded from these studies that collagenase and the gelatinases play a role in the tissue remodeling associated with luteal development, proteoglycanase is associated with luteal maintenance, and collagenase may contribute to the structural regression of the corpus luteum.

Hormonal regulation of tissue inhibitors of metalloproteinases during follicular development in the rat ovary.

Tissue inhibitors of metalloproteinases (TIMPs) are membres of a multigene family of proteinase inhibitors that regulate the activity of metalloproteinases. To test the hypothesis that TIMPs regulate connective tissue remodeling during follicular development, rats were injected with PMSG (20 IU, sc), and ovaries and serum were collected at the time of pregnant mare serum gonadotropin at the time of pregnant mare serum gonadotropin (PMSG) administration (0 h) and at 6, 12, 24, 36, and 48 h later for analysis of TIMP expression, metalloproteinase inhibitor activity, and steroidogenesis. Serum estradiol levels increased from 20.9 pg/mL at 0 h to 461 pg/mL at 48 h. Northern analysis was performed for analysis of TIMP-1, TIMP-2, and TIMP-3 expression (N = 4). For TIMP-1, PMSG stimulated a 2.4- to 2.5-fold increase in TIMP-1 mRNA at 6 and 12 h compared to ovaries collected at the time of PMSG administration (i.e., 0 h control). TIMP-1 mRNA returned to control levels within 24 h and remained unchanged through 48 h. In contrast to TIMP-1, TIMP-3 mRNA decreased by approx 2.5-fold at 6 h following PMSG administration, and expression remained decreased through 48 h. For TIMP-2, the expression of the 3.5-kb transcript decreased at 24 h after PMSG, whereas expression of the 1 kb transcript was unchanged. There was no change in metalloproteinase inhibitor activity in whole ovarian extracts between 0 and 36 h. However, there was an increase in inhibitor activity at 48 h. These findings are the first demonstration of hormonal regulation of TIMPs during the follicular phase. The differential regulation of the TIMPs by gonadotropins, for example, an increase in TIMP-1 and a concomitant decrease in TIMP-3 expression, may reflect different roles, sites of action, or enzyme specificity for the inhibitors as the follicle grows.

Galanin: a novel intraovarian regulatory peptide.

Galanin is a 29-amino acid peptide that acts as a neuropeptide in many tissues. To date, galanin action and the hormonal regulation of galanin gene expression have not been described in the ovary of any species. To study possible ovarian expression and regulation of galanin, immature gonadotropin-primed rats were given hCG (10 IU), and their ovaries were collected 0, 4, 8, 12, and 20 h after hCG treatment for determination of galanin messenger RNA (mRNA) concentration by solution hybridization. Galanin mRNA levels progressively increased after hCG administration, peaking at 12 h (2.4-fold increase vs. 0 h), with a subsequent return to 0 h levels at 20 h. To determine a possible ovarian role for galanin, rats were killed 48 h after gonadotropin administration, their ovaries were removed, and granulosa cells were harvested. These cells and the ovarian tissue remaining after granulosa cell collection (i.e. "shells") were each cultured for 24 h with increasing concentrations of galanin (0, 10, 100, and 1000 nM) in the presence or absence of LH. The medium was examined for steroid production and metalloproteinase inhibitor activity. In granulosa cell cultures, galanin increased the levels of estradiol by 26% and had no effect on progesterone, but decreased metalloproteinase inhibitor activity by 61% in the conditioned medium. In the shell cultures, galanin increased estradiol, progesterone, and androstenedione in the medium, suggesting that galanin acts on cells other than granulosa cells or that galanin action requires a paracrine interaction between granulosa and thecal cells. Our data demonstrate that galanin message is increased by hCG, and that galanin acts to amplify ovarian steroidogenesis while decreasing metalloproteinase inhibitor activity. These findings establish that ovarian galanin mRNA is hormonally stimulated and that galanin acts as an intraovarian regulatory peptide.

Antiprogesterone (RU486) effects on metalloproteinase inhibitor activity in human and rat granulosa cells.

OBJECTIVE: To determine if granulosa cells are a source of metalloproteinase inhibitor activity and whether P regulates this activity. DESIGN: Inhibitor activity was measured in media from human and rat granulosa cells cultured with the antiprogesterone mifepristone (RU486). Human granulosa cells were obtained at the time of oocyte retrieval from gonadotropin-stimulated patients after hCG administration. Rat cells were collected from gonadotropin-primed animals before the LH surge. Human and rat cells were cultured for 24 hours in the absence or presence of LH (100 ng/mL or 3.3 nmol/L) and/or RU486 (5 microM or 50 microM). Inhibitor activity and P were measured in the media. SETTING: Reproductive Endocrinology Laboratories, University of Kentucky. RESULTS: Media from human granulosa cells contained metalloproteinase inhibitor activity and the addition of LH did not change this activity. RU486 at 50 microM decreased inhibitor activity in cells cultured in the absence or presence of LH (0.59 +/- 0.12- and 0.24 +/- 0.18-fold change, respectively, versus control cultures; mean +/- SEM). In rat granulosa cells, inhibitor activity increased with LH treatment (1.97 +/- 0.12-fold change). RU486 decreased the activity present in cells cultured in the presence of LH. Progesterone production was stimulated by LH in both human and rat granulosa cells (3.71 +/- 0.90- and 7.18 +/- 0.24-fold change, respectively). In the human cells, RU486 inhibited P production whereas RU486 stimulated P production in the rat cells. CONCLUSIONS: These findings demonstrate for the first time that human granulosa cells are a source of metalloproteinase inhibitor activity. The decrease in granulosa cell-derived inhibitor activity by RU486 suggests that P stimulates inhibitor activity. Thus, P may regulate proteolysis associated with follicular rupture via its ability to stimulate granulosa cell production of metalloproteinase inhibitors. Differences in P production between the human and rat cells may be due to differences in hormonal stimulation regimens (i.e., hCG exposure).

Gelatinase and proteoglycanase activity during the periovulatory period in the rat.

Gelatinase and proteoglycanase are metalloproteinases that govern extracellular matrix remodeling. In the present study, immature rats were primed with eCG (20 IU) and hCG (10 IU). Ovarian gelatinase and proteoglycanase activity were determined at the time of hCG administration (0 h) as well as 4, 8, and 12 h later. Gelatinase and proteoglycanase were extracted by homogenization in Triton and by heating (i.e., heat extraction). An aliquot of the heat extract was reduced and alkylated to destroy metalloproteinase inhibitors. Heat extracts not reduced and alkylated showed low levels of gelatinase and proteoglycanase activity that did not change at the different time points. However, with reduction and alkylation, gelatinolysis increased approximately 4-fold (p less than 0.05) at 4 h, 8 h, and 12 h after hCG priming. Proteoglycanase activity increased approximately 2-fold (p less than 0.05) between 0 and 8 h and declined at 12 h after hCG. The ovarian gelatinolytic activity was due to a metalloproteinase as demonstrated by the inhibition of enzyme activity by phenanthroline and EDTA (97.1 +/- 0.7% and 97.4 +/- 0.6% inhibition respectively). Proteoglycanase activity was not inhibited by phenanthroline (11.5 +/- 3.5%), suggesting that the enzyme activity was not specifically a metal-dependent enzyme. Gelatin gel zymography of the ovarian extracts demonstrated four predominant and distinct gelatin-degrading enzymes of 78, 72, 66, and 62 kDa, similar to the size of gelatinase. The present findings demonstrate a periovulatory increase in ovarian gelatinolytic and proteglycanase activity that may play a pivotal role in connective tissue remodeling associated with ovulation.

Characterisation of the peanut lectin-binding glycoproteins of human epidermal keratinocytes.

We have previously shown that peanut lectin (PNA) binding is a useful marker of keratinocyte terminal differentiation and have identified two PNA-binding glycoproteins with electrophoretic mobilities of approximately 250 kDa and 110 kDa [11]. We now report that in epidermis and stratified cultures of keratinocytes the binding patterns of PNA and the sialic acid-specific lectin Limax flavus agglutinin (LFA) are complementary, with LFA showing specificity for cells in the basal layer. LFA bound to the 250-kDa glycoprotein immunoprecipitated with an antiserum raised against the PNA-binding glycoproteins (anti PNA-gp), but not to the 110-kDa glycoprotein; it also bound additional high-molecular-weight material. These data suggest that the 250-kDa glycoprotein is expressed in the basal layer in a form with terminal sialic acid residues and suprabasally in a form with terminal galactose. LFA and anti-PNA-gp stained all cells in a range of cultured epithelial lines tested, whereas PNA stained only cells that had lost contact with the culture substratum, raising the possibility that loss of sialic acid residues is associated with stratification. Anti PNA-gp recognized glycoproteins of differing mobilities in these lines. Anti PNA-gp also stained epithelial cells in all tissues tested. In keratinocytes the PNA-binding glycoproteins were localised to the cell surface by immunoelectron microscopy; they were abundant on the microvilli and absent from desmosomal junctions. In conclusion, we have obtained further information about the nature of the PNA-binding glycoproteins in keratinocytes and related glycoproteins in other epithelial cell types.

Onset of expression of peanut lectin-binding glycoproteins is correlated with stratification of keratinocytes during human epidermal development in vivo and in vitro.

During gestation the epidermis develops from a single layer of ectoderm into a layer of keratinocytes overlaid by a layer of periderm; this is followed by a progressive increase in the number of layers of keratinocytes, until finally the distinct granular and cornified layers characteristic of mature epidermis are formed. As part of our investigation into the function of the peanut lectin-binding glycoproteins of cultured human keratinocytes, we have examined their expression at different stages of human epidermal development. We found that the onset of expression of the glycoproteins coincided with the transition from a two- to a three-layered epidermis, both in vivo and in organ culture. In adult epidermis, the patterns of binding of peanut lectin and Limax flavus lectin are complementary, with peanut binding more strongly to suprabasal keratinocytes and Limax flavus lectin binding more strongly to cells in the basal layer. We found that the complementary pattern of binding of the two lectins was established at, or shortly after, the onset of stratification and retained throughout development. In contrast, expression by keratinocytes of involucrin, a protein precursor of the cornified envelope, occurred after stratification had begun. Finally, we identified the peanut lectin-binding glycoproteins of adult epidermis by immunoblotting with an antiserum raised against the glycoproteins of cultured neonatal keratinocytes. In conclusion, expression of the peanut lectin-binding glycoproteins is an early event in epidermal development, and this would be consistent with a role for the glycoproteins in stratification.

The relationship among retinoid structure, affinity for retinoic acid-binding protein, and ability to respecify pattern in the regenerating axolotl limb.

To further our understanding of the action of retinoids on the respecification of pattern in the regenerating axolotl limb we have studied the relative potencies of a range of synthetic and natural retinoids administered locally to the blastema. Alterations in the polar end group of the retinoic acid (RA) molecule to produce esters, the alcohol, or the aldehyde abolish the ability of the molecule to respecify pattern. On the other hand, alterations of the ring or side chain to produce the synthetic retinoids arotinoid and TTNPB considerably increases the potency of the molecule to respecify pattern--TTNPB is at least 100X more potent than retinoic acid. To examine the role of cellular retinoic acid-binding protein (CRABP) in the respecification process we determined the relative binding affinities of these retinoids for CRABP. These data correlated well with the respecification series: retinoids which showed no affinity for CRABP did not respecify pattern and those which did show affinity for CRABP did respecify pattern. Furthermore the most potent retinoid, TTNPB, has a higher affinity for CRABP than RA itself. This suggests that CRABP may be playing an important role in the action of RA on pattern formation in the regenerating limb.

The peanut lectin-binding glycoproteins of human epidermal keratinocytes.

Peanut lectin (PNA) is known to bind more strongly to keratinocytes that are undergoing terminal differentiation than to proliferating keratinocytes, both in intact epidermis and in culture. In order to investigate the significance of this change in cell-surface carbohydrate we have identified the PNA-binding glycoproteins of cultured human keratinocytes and raised antibodies against them. Two heavily glycosylated bands of 110 and 250 kDa were resolved by PAGE of [14C]galactose- or [14C]mannose- and [14C]glucosamine-labeled cell extracts eluted with galactose from PNA affinity columns. The higher molecular weight band was also detected on PNA blots of unlabeled cell extracts transferred to nitrocellulose. Both bands were sensitive to Pronase digestion, but only the 250-kDa band was digested with trypsin. A rabbit antiserum that we prepared (anti-PNA-gp) immunoprecipitated both bands from cell extracts. In contrast to PNA, anti-PNA-gp bound equally to proliferating and terminally differentiating cells, indicating that some epitope(s) of the PNA-binding glycoproteins is present on the cell surface prior to terminal differentiation. When keratinocytes grown as a monolayer in low-calcium medium (0.1 mM calcium ions) were switched to medium containing 2 mM calcium ions in order to induce desmosome formation and stratification, there was a dramatic redistribution of the PNA-binding glycoproteins, which became concentrated at the boundaries between cells. This may suggest a role for the glycoproteins in cell-cell interactions during stratification.

The role of cartilage and fibronectin during respecification of pattern induced in the regenerating amphibian limb by retinoic acid.

When retinoic acid (RA) is applied to the regenerating limb the positional information of blastemal cells is respecified and extra limb segments develop. We are trying to elucidate the molecular basis of the action of RA and report here experiments focused on the role that fibronectin (FN) might play in the process. The FN distribution in stump tissues, regeneration blastemas and RA-treated blastemas was investigated by immunocytochemistry. Two effects of RA were observed. Firstly, excessive dedifferentiation of the severed cartilage at the amputation plane, resulting in lumps of FN-positive matrix being released into the blastema; secondly, blastemal cells tend to aggregate together into FN-positive accumulations. Excessive dedifferentiation of the cartilage plays no role in the RA-induced respecification of pattern, because we show that extra segments are still produced in RA-treated limbs from which all the cartilage has been removed. The effect on blastemal cell FN distribution was investigated in several ways. Axolotl plasma FN and cellular FN were characterised on immunoblots, and no obvious change was observed after RA treatment; neither were there changes in amounts of FN detected by ELISA. Levels of FN synthesis were measured by [35S]-methionine labelling and again no change observed after RA treatment. We conclude that the change in FN distribution observed by immunocytochemistry after RA treatment may be due to the retention of FN on the surface of the blastemal cells rather than to any effect on the levels of synthesis of this molecule.

Retinoic acid-binding protein in the axolotl: distribution in mature tissues and time of appearance during limb regeneration.

Analysis of cytoplasmic protein preparations from axolotl tissues revealed the presence of a cytoplasmic retinoic acid-binding protein (CRABP), of approximate molecular weight 17K. This protein was found to be present at various concentrations in skin, muscle, and limb tissue preparations, but not in liver and serum preparations. The distribution and molecular weight of this protein agrees with that reported in mammalian studies. The level of CRABP in cone stage blastemas was found to be significantly higher than that found in nonregenerating whole limb preparations. The level falls gradually, to approach normal, towards the completion of regeneration. Such an increase, at the start of regeneration, was not altered by 4 days pretreatment with 36 mg/liter all-trans-retinoic acid, a sufficient dose to produce pattern effects. Competition experiments confirmed that the all-trans and 13-cis isomers of retinoic acid bind to CRABP with similar high efficiencies, and that the arotinoid, Ro 13-6298, exhibits only a fraction of this binding activity. Retinol, retinol palmitate, and retinol acetate were unable to compete with [3H]retinoic acid for binding to CRABP. The results presented here are discussed in terms of their possible value to understanding pattern specification in the regenerating urodele limb.

Role of the infraorbital nerve in retrieving behavior in lactating rats.

Injecting the local lidocaine into the mystacial pads of primiparous rats rendered the snout insensitive to touch and abolished the dams' ability to retrieve. Injecting the drug into the masseter muscles or intraperitoneally did not render the snout anaptic or abolish retrieval, results indicating that the effect of intramystacial lidocaine treatment could not be attributed to systemic toxicity or to the drug's spreading from the mystacial pads to affect the nearby masseter muscles. Cutting the intraorbital nerves produced a temporary retrieval impairment that was indistinguishable from that produced by intramystacial lidocaine injection. Surgical deafferentation did not affect the latency of dams to approach pups that had been displaced from the nest site, which indicates that the retrieval deficit was not due to postoperative debilitation. In addition, infraorbital section did not interfere with the ability to locate and consume a piece of cheese that had been buried in the home cage, which indicates that the operation did not produce anosmia or interfere with the muscles used to grasp pups. Cutting the facial nerves abolished vibrissal movement but did not disrupt retrieval, results indicating that the effect of infraorbital lesions could not be attributed to the loss of vibrissal cues or to nonspecific effects of nerve section. The possibility was tested that infraorbital deafferentation has a profound effect on retrieval because anaptic dams, in their initial attempts at picking up pups, elicit distress vocalizations from their offspring. Dams injected with lidocaine in the mystacial pads failed to retrieve pups that had been anesthetized with a barbiturate to abolish their distress vocalizations. Thus pup-produced vocalizations are not responsible for the retrieval impairment exhibited by anaptic mothers. It is concluded that perioral tactile sensation plays an important role in the ability of lactating rats to retrieve.

The role of perioral sensation in nipple attachment by weanling rat pups.

Injecting .05 ml of 1% lidocaine into each vibrissal pad, or cutting the infraorbital nerves, abolished nipple attachment in weanling Wistar rat pups. Nipple attachment recovered following infraorbital section. Injecting the local anesthetic intraperitoneally, or into the region of the masseter muscles, did not disrupt attachment, indicating that the effect of the drug on suckling was specific to the site of injection and could not be attributed to systemic toxicity or paralysis of the masseter muscles. Performance on an olfactory-guided orientation task was not disrupted by lidocaine, indicating that the drug did not render pups anosmic. Tactile sensation in the vibrissal pads, rhinarium, and upper lip was abolished after injecting the drug into the vibrissal pads. Vibrissal movement was absent following injection of lidocaine into either the vibrissal pads or the region of the masseter muscles. Shaving the vibrissae did not disrupt nipple attachment. The results are interpreted as suggesting that the nipples' textural qualities elicit attachment in weanling pups.